What is the substance that tumors release that stimulates growth of blood vessels but suppresses its release from other tumors?

What is the substance that tumors release that stimulates growth of blood vessels but suppresses its release from other tumors?

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I'm currently in high school and I am working on a cancer research project. My project consists of a cancer, and different ways to treat it. I have a set of benign tumor and I was thinking of immediate way of treating it without using a drug like Taxol to destroy parts of the cell is to simply remove the tumor.

However, I remembered learning about a 1 1/2 years ago about a substance that tumors release to stimulate the growth of blood vessels towards them, but this same substance suppresses the release of itself by another tumor (hopefully this makes some sense). I made some drawings that are attached here:

Scenario 1: The 1st tumor in the blood flow releases an unknown substance, which promotes blood vessel growth, but suppresses the release of the same substance from another tumor behind it.

Scenario 2: The 1st tumor is removed, and now the 2nd tumor starts to release the substance, because the 1st tumor's release is not suppressing the 2nd tumor's release anymore.

Does anyone know the name of this substance, and can someone help me learn more about it? Thanks.

One very important signal for angiogenesis are the Vascular endothelial growth factors (VEGF) (a small group of ~5 similar proteins). They generally promote generation of new blood vessels & migration of these towards the source of VEGF expression.
Blocking VEGF signalling (mostly be blocking the receptors with antibodies) has been a pretty good strategy in cancer treatment, since tumors can't grow over a certain size without blood supply by new grown vessels.

However, I am not aware of any blood-vessel specific signalling mechanism that stops it's own expression at a secondary site. (This may be possible though, especially for specific signals)

Galectins are family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. These proteins are produced by tumor cells and promote angiogenesis.

These secreted Galectins are used by endothelial cells to enhance migration and proliferation. And play various roles during development of tumor such as cellular adhesion, cell mobility, tumor-induced angiogenesis, and apoptosis.



When a tumor stimulates the growth of new vessels, it is said to have undergone an 'angiogenic switch'. The principal stimulus for this angiogenic switch appears to be oxygen deprivation, although other stimuli such as inflammation, oncogenic mutations and mechanical stress may also play a role. The angiogenic switch leads to tumor expression of pro-angiogenic factors and increased tumor vascularization. [4] Specifically, tumor cells release various pro-angiogenic paracrine factors (including angiogenin, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor-β (TGF-β). These stimulate endothelial cell proliferation, migration and invasion resulting in new vascular structures sprouting from nearby blood vessels. Cell adhesion molecules, such as integrins, are critical to the attachment and migration of endothelial cells to the extracellular matrix. [4]

VEGF pathway inhibition Edit

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. Strategies to inhibit the VEGF pathway include antibodies directed against VEGF or VEGFR, soluble VEGFR/VEGFR hybrids, and tyrosine kinase inhibitors. [4] [5] The most widely used VEGF pathway inhibitor on the market today is Bevacizumab. [ citation needed ] Bevacizumab binds to VEGF and inhibits it from binding to VEGF receptors. [6]

Angiogenesis is regulated by the activity of endogenous stimulators and inhibitors. Endogenous inhibitors, found in the body naturally, are involved in the day-to-day process of regulating blood vessel formation. Endogenous inhibitors are often derived from the extracellular matrix or basement membrane proteins and function by interfering with endothelial cell formation and migration, endothelial tube morphogenesis, and down-regulation of genes expressed in endothelial cells.

During tumor growth, the action of angiogenesis stimulators surpasses the control of angiogenesis inhibitors, allowing for unregulated or less regulated blood vessel growth and formation. [7] Endogenous inhibitors are attractive targets for cancer therapy because they are less toxic and less likely to lead to drug resistance than some exogenous inhibitors. [4] [5] However, the therapeutic use of endogenous inhibitors has disadvantages. In animal studies, high doses of inhibitors were required to prevent tumor growth and the use of endogenous inhibitors would likely be long-term. [7]

Inhibitors Mechanism
soluble VEGFR-1 and NRP-1 decoy receptors [8] for VEGF-B and PIGF
Angiopoietin 2 antagonist of angiopoietin 1
TSP-1 and TSP-2 inhibit cell migration, cell proliferation, cell adhesion and survival of endothelial cells
angiostatin and related molecules inhibit cell proliferation and induce apoptosis of endothelial cells
endostatin inhibit cell migration, cell proliferation and survival of endothelial cells
vasostatin, calreticulin inhibit cell proliferation of endothelial cells
platelet factor-4 inhibits binding of bFGF and VEGF
TIMP and CDAI inhibit cell migration of endothelial cells
Meth-1 and Meth-2
IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18 inhibit cell migration of endothelial cells, downregulate bFGF
prothrombin (kringle domain-2), antithrombin III fragment inhibit cell proliferation of endothelial cells
prolactin VEGF
VEGI affects cell proliferation of endothelial cells
SPARC inhibit binding and activity of VEGF
osteopontin inhibit integrin signalling
maspin inhibits proteases
canstatin (a fragment of COL4A2) inhibits endothelial cell migration, induces apoptosis [9]
proliferin-related protein mannose 6-phosphate binding lysosomal protein [10]

A recent method for the delivery of anti-angiogenesis factors to tumor regions in cancer patients uses genetically modified bacteria that are able to colonize solid tumors in vivo, such as Clostridium, Bifidobacteria and Salmonella by adding 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 from being able to gain access to oxygen and ultimately starving the cancer cells. [11] This method has been shown to work both in vitro and in vivo in mice models, with very promising results. [12] It is expected that this method will become commonplace for treatment of various cancer types in humans in the future. [ citation needed ]

Diet Edit

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:

    such as tofu and tempeh, (which contain the inhibitor "genistein") [13]
  • Agaricus subrufescens mushrooms (contain the inhibitors sodium pyroglutamate and ergosterol) [14][15] (Rubus occidentalis) extract [16] (via inhibition of VEGF and TGF-beta) [17]
  • Trametes versicolor mushrooms (Polysaccharide-K) [18][19][20]
  • Maitake mushrooms (via inhibition of VEGF) [21]
  • Phellinus linteus mushrooms [22] (via active substance Interfungins A inhibition of glycation) [23] (catechins) [24] (glycyrrhizic acid) [25] (resveratrol) [25]
  • Antiangiogenic phytochemicals and medicinal herbs [26] (Queen bee acid) [27]

Drugs Edit

Research and development in this field has been driven largely by the desire to find better cancer treatments. Tumors cannot grow larger than 2mm without angiogenesis. By stopping the growth of blood vessels, scientists hope to cut the means by which tumors can nourish themselves and thus metastasize.

In addition to their use as anti-cancer drugs, angiogenesis inhibitors are being investigated for their use as anti-obesity agents, as blood vessels in adipose tissue never fully mature, and are thus destroyed by angiogenesis inhibitors. [28] Angiogenesis inhibitors are also used as treatment for the wet form of macular degeneration. By blocking VEGF, inhibitors can cause regression of the abnormal blood vessels in the retina and improve vision when injected directly into the vitreous humor of the eye. [29]

Overview Edit

Inhibitors Mechanism
bevacizumab (Avastin) VEGF
itraconazole inhibits VEGFR phosphorylation, glycosylation, mTOR signaling, endothelial cell proliferation, cell migration, lumen formation, and tumor associated angiogenesis. [30] [31] [32]
carboxyamidotriazole inhibit cell proliferation and cell migration of endothelial cells
TNP-470 (an analog of fumagillin)
CM101 activate immune system
IFN-α downregulate angiogenesis stimulators and inhibit cell migration of endothelial cells
IL-12 stimulate angiogenesis inhibitor formation
platelet factor-4 inhibits binding of angiogenesis stimulators
VEGFR antagonists
angiostatic steroids + heparin inhibit basement membrane degradation
Cartilage-Derived Angiogenesis Inhibitory Factor
matrix metalloproteinase inhibitors
angiostatin inhibit cell proliferation and induce apoptosis of endothelial cells
endostatin inhibit cell migration, cell proliferation and survival of endothelial cells
2-methoxyestradiol inhibit cell proliferation and cell migration and induce apoptosis of endothelial cells
tecogalan inhibit cell proliferation of endothelial cells
tetrathiomolybdate copper chelation which inhibits blood vessel growth
thalidomide inhibit cell proliferation of endothelial cells
thrombospondin inhibit cell migration, cell proliferation, cell adhesion and survival of endothelial cells
prolactin VEGF
αVβ3 inhibitors induce apoptosis of endothelial cells
linomide inhibit cell migration of endothelial cells
ramucirumab inhibition of VEGFR2 [33]
tasquinimod Unknown [34]
ranibizumab VEGF [35]
sorafenib (Nexavar) inhibit kinases
sunitinib (Sutent)
pazopanib (Votrient)
everolimus (Afinitor)

Bevacizumab Edit

Through binding to VEGFR and other VEGF receptors in endothelial cells, VEGF can trigger multiple cellular responses like promoting cell survival, preventing apoptosis, and remodeling cytoskeleton, all of which promote angiogenesis. Bevacizumab (brand name Avastin) traps VEGF in the blood, lowering the binding of VEGF to its receptors. This results in reduced activation of the angiogenesis pathway, thus inhibiting new blood vessel formation in tumors. [7]

After a series of clinical trials in 2004, Avastin was approved by the FDA, becoming the first commercially available anti-angiogenesis drug. FDA approval of Avastin for breast cancer treatment was later revoked on November 18, 2011. [36]

Thalidomide Edit

Despite the therapeutic potential of anti-angiogenesis drugs, they can also be harmful when used inappropriately. Thalidomide is one such antiangiogenic agent. Thalidomide was given to pregnant women to treat nausea. However, when pregnant women take an antiangiogenic agent, the developing fetus will not form blood vessels properly, thereby preventing the proper development of fetal limbs and circulatory systems. In the late 1950s and early 1960s, thousands of children were born with deformities, most notably phocomelia, as a consequence of thalidomide use. [37]

Cannabinoids Edit

According to a study published in the August 15, 2004 issue of the journal Cancer Research, cannabinoids, the active ingredients in marijuana, restrict the sprouting of blood vessels to gliomas (brain tumors) implanted under the skin of mice, by inhibiting the expression of genes needed for the production of vascular endothelial growth factor (VEGF). [38]

General side effects of drugs Edit

Bleeding Edit

Bleeding is one of the most difficult side effects to manage this complication is somewhat inherent to the effectiveness of the drug. Bevacizumab has been shown to be the drug most likely to cause bleeding complications. [ citation needed ] While the mechanisms of bleeding induced by anti-VEGF agents are complicated and not yet totally understood, the most accepted hypothesis is that VEGF could promote endothelial cell survival and integrity in the adult vasculature and its inhibition may decrease capacity for renewal of damaged endothelial cells. [39]

Increased blood pressure Edit

In a study done by ML Maitland, a mean blood pressure increase of 8.2 mm Hg systolic and 6.5 mm Hg diastolic was reported in the first 24 hours after the first treatment with sorafenib, a VEGF pathway inhibitor. [40] [ non-primary source needed ]

Less common side effects Edit

Because these drugs act on parts of the blood and blood vessels, they tend to have side effects that affect these processes. Aside from problems with hemorrhage and hypertension, less common side effects of these drugs include dry, itchy skin, hand-foot syndrome (tender, thickened areas on the skin, sometimes with blisters on palms and soles), diarrhea, fatigue, and low blood counts. Angiogenesis inhibitors can also interfere with wound healing and cause cuts to re-open or bleed. Rarely, perforations (holes) in the intestines can occur. [39]

By Yeong Sek Yee And Khadijah Shaari

The concept of angiogenesis is very new. It was only in 1994 that, after Dr Judah Folkman’s key concept of his new theory of cancer was published in the periodical “CELL” that overnight, angiogenesis became one of the principal targets in cancer research. What then is angiogenesis?

Briefly angiogenesis means blood vessel formation. Tumour angiogenesis is the growth of new blood vessels that tumours need to grow and this is caused by the release of chemicals by the tumour. Conversely, angiogenesis inhibitor is a substance that may prevent the formation of blood vessels. In anti-cancer therapy, an angiogenesis inhibitor may prevent the growth of new blood vessels that tumours need to grow.

In “ANTICANCER: A NEW WAY OF LIFE,” Dr David Servan-Schreiber, a clinical professor of psychiatry at the University of Pittsburgh School of Medicine, described Dr Judah Folkman’s various experiments in the late 1960s and 1970s that gave him (Dr Folkman) the first glimmering of a wild inspired hunch. What if cancerous tumours, in order to expand, needed to trigger the growth of new blood vessels to feed themselves? And if that was true, what if a way could be found to stop that growth? Could cancers be starved to death? Experiment by experiment, Dr Folkman built up the key concepts of his new theory of cancer (i.e. angiogenesis). Some main points of Dr Folkman’s theory (see page 52 of ANTICANCER) are:

  • Micro tumours cannot change into dangerous cancers without creating a new network of blood vessels to feed them.
  • To do so, they produce a chemical substance called angiogenin that forces the vessels to approach them and to sprout new branches.
  • The new tumour cells that spread to the rest of the body i.e. metastasis are dangerous only when they are able, in turn, to attract new blood vessels.
  • Large primary tumours send out metastases….but as in any colonial empire, they prevent these distant territories from becoming too important by producing another chemical substance that block the growth of new blood vessels – angiostatin.(This explains why metastases sometimes suddenly grow once the principal tumour has been surgically removed)

Dr Folkman spent 20 years in the wilderness. Nobody believed him. He was scorned, criticised and described as a looney. Other doctors shook their heads at the waste of a great mind, and ambitious young medical researchers were told that accepting a position in Folkman’s lab would be the death of their careers. In “ANTICANCER,” Dr Schreiber described Dr Folkman’s 20 years journey in the wilderness as “Crossing the Dessert” (page 53). This is a classic example of Schopenhauer’s saying:–All great truth goes through three phases. First, it is ridiculed, then violently attacked, and finally accepted as self-evident(page 53). This will probably be the case in the concept of anti-angiogenic foods as described in the ensuing sections.

(NB: Perhaps, if you would like to follow Dr Folkman’s journey “Crossing the Desert,” do read “DR FOLKMAN’S WAR” written by acclaimed science writer Robert Cooke. Reading the forward by Dr Everett Koop, MD, ScD, you will soon realise that the title of the book is not Dr Folkman’s War against cancer but it was a war against the scientific and medical community which took more than 20 years to recognise his concept of angiogenesis).

Today, many drugs similar to angiostatin (such as Avastin, Sutent and Nexavar) have been developed by the pharmaceutical industry. But “their effect on humans when used alone have turned out to be disappointing” (ANTICANCER page 54). This view is also shared by medical oncologist Dr Richard Frank, MD (in FIGHTING CANCER WITH KNOWLEDGE AND HOPE) in which he said that…“although targeted therapies (angiogenesis inhibitor drugs as mentioned above) were developed with the hope that they would be magic bullets that would neatly eradicate cancer through the selective targeting of one critical molecule, in general they have fallen short of their lofty goal” (page180). Anti-angiogenesis drugs have produced more troublesome side effects than foreseen. As a result, they are probably not the long-hoped-for miracle drugs (ANTICANCER page 54).

According to Dr David Servan-Schreiber, as an alternative to waiting for the miracle drug, there are natural approaches that have a powerful effect on angiogenesis without side effects and that can be combined perfectly with conventional treatments (page54). These are:

  • Specific dietary practices (many natural anti-angiogenesis foods have been discovered recently, including common edible mushrooms, green tea, spices, and herbs)..
  • Everything that contributes to reducing inflammation, the direct cause of the growth of new blood vessels.

Anti-angiogenesis foods listed by Dr Schreiber are green tea, olives and olive oil, turmeric and curry, ginger, cruciform vegetables, garlic, onion, leeks, shallots, chives, vegetables and fruits rich in carotenoids, tomatoes and tomato sauce, soy, mushrooms, herbs, and spices, seaweed, berries, plums, peaches & nectarines, citrus fruits, pomegranate juice, red wine, dark chocolate, vitamin D, Omega-3s, probiotics and foods rich in selenium. (For a complete exposure of these foods we urge you to read Chapter 8: The Anti-Cancer Foods . We also urge you to watch the DVD entitled “AntiCancer with Dr David Servan-Schreiber.” Some links are available on as follows:

a) Dr David Servan-Schreiber’s Remarkable Story:

b) Natural Defences in Preventing and Treating Cancer:

Anti-angiogenis or anti-angiogenic foods?Your doctor/ oncologist will in all probability pour scorn on this concept with the usual comments–not proven, not scientifically tested, etc. But frankly, are all the conventional cancer treatments properly and scientifically and independently tested?

Who else has done research and written about anti-angiogenic dietary factors under the concept of angiogenesis?

In the forefront of such research is Dr William Li MD, the founder of The Angiogenesis Foundation, the world’s first non-profit organisation dedicated to conquering disease using the new approach based on angiogenesis, the growth of new capillary blood vessels in the body.

According to Dr Li, many foods contain naturally occurring inhibitors of angiogenesis. When these foods are consumed and absorbed into the blood stream, the inhibitors act to boost the body’s existing system that suppresses undesirable angiogenesis that can promote or accompany disease.

The following is a list of foods (according to Dr Li) that have innate properties which inhibit angiogenesis, thus working to cut off cancer tumours from blood supplies. These are green tea, berries, citrus fruits, apples, pineapple, cherries, red grapes, red wine, cruciferous vegetables, soybeans, ginseng, mushroom, liquorice, turmeric, nutmeg, lavender, artichokes, pumpkin, sea cucumber, tuna, parsley, garlic, tomato, olive oil, grape seed oil, dark chocolate. (Source: Angiogenesis Foundation Website:

Also we recommend that you watch a video of Dr William Li enlightening you about “angiogenesis,” its impact on the human body, its connection to cancer and how you can deal with it.

To view the video, try the following links: –

Dr Judah Folkman’s visionary ideas on cancer treatment served as a starting point and inspired two Canadian cancer researchers to theorise and confirm that “there is some weakness in the armor of tumor cells that might allow us to better our chances of destroying them” (Incidentally Chapter 4 in Dr Schreiber book “ANTICANCER” is entitled “Cancer’s Weakness”) These two researchers Dr Richard Beliveau, PhD and Dr Denis Gingras, PhD worked on the premise that “despite its great power, its versatility, and its enormous ability to adapt to hostile conditions of neighbouring cells, the cancer cells remains extremely dependent upon its energy needs. To grow, a tumour requires a constant supply of oxygen and nutrients. Their studies strongly suggest that certain types of cancers can be prevented by modifying our dietary habits to include foods with the power to fight tumours at the source and thus prevent their growth.

According to Dr Believeau and Dr Gingras, “nature supplies us with an abundance of foods rich in molecules with very powerful anticancer properties capable of engaging with the disease without causing any harmful side effects. In many respects, these foods possess therapeutic properties on par with those of synthetic drugs” (Ha, Big Pharma definitely won’t like this statement)

Some of the specific foods researched by Dr Beliveau and Dr Gingras are: cruciferous vegetables, garlic and onions, soy, turmeric, green tea, berries, omegs-3s, tomatoes, fresh fruits, and dark chocolates.

Dr Beliveau and Dr Gingras distilled their research findings into a simple book for the lay person- “FOODS TO FIGHT CANCER” –the goal of this book is to present a summary of the scientific studies currently available.

Another medical doctor who believes and has written on the subject of angiogenesis is Dr Joel Fuhrman, a board-certified family physician who specializes in preventing and reversing disease through nutritional and natural methods. In this book “SUPER IMMUNITY” Dr Fuhrman touched on angiogenesis in Chapter 3 under the heading, “The Anticancer Solution”The salient points in this section are: –

  • Many plant foods contain natural angiogenesis inhibitors- especially mushrooms
  • Dietary angiogenesis inhibitors are now being investigated as a preventive strategy to “starve” cancers while they are still small and harmless.
  • If our diet contains plenty of angiogenesis inhibitors, it can prevent small tumours from acquiring a blood supply and growing larger and becoming more aggressive or cancerous.
  • Some anti-angiogenic foods/nutrients listed by Dr Fuhrman are allium vegetables, berries, black rice, cinnamon, citrus fruits, cruciferous vegetables, flax seeds, ginger, Grapes, green tea, mushrooms, Omega-3 fats, peppers, pomegranate, quince, resveratrol, soybeans, spinach, tomatoes, and turmeric. (Scientific studies are quoted by Dr Fuhiman in the end NOTES)
  • On the other hand, “there are foods and nutrients that promote angiogenesis–and thus obesity and cancer. These include white-flour based breads and sweets that raise insulin levels, and the high-fat, high-cholesterol, standard, Western diet. These modern, unhealthy foods promote fat storage in addition to having a high-caloric density. They are a double negative, while green, mushrooms, onions, berries and the other foods listed above are a double positive”

In concluding the chapter, Dr Fuhrman laments that… “many people choose to reject new science even when the evidence is overwhelming. This book, SUPER IMMUNITY, may be attacked by people in powerful positions of authority whose livelihood is dependent on competing interests such as “recreational” foods, drugs and medical technology. Does this sound familiar to you?

In “FIGHTING CANCER WITH KNOWLEDGE AND HOPE,” oncologist Dr Richard Frank clearly stressed that:

  • Diet can promote or inhibit the formation of cancer in many ways
  • There are both good and bad foods to influencing the development of cancer
  • More direct links between particular components of food and cancer have been confirmed by some recent studies. A classic link is attached.

Although “anti-angiogenesis drugs (like Avastin, Sutent, Nexavar) prevent tumours from growing the blood vessels they need to grow, none is perfect” (page 481). This is the view of Dr Keith Block, MD an Integrative Oncologist who explained that “just as tumours can switch to a second growth pathway if their primary pathway is blocked by a chemotherapy drug, so tumour can switch to a backup pathway for growing blood vessels when the first pathway is blocked by an anti-angiogenesis drug”(page 481).

Just as drug cocktails are a hot area of research in mainstream oncology, so combinations of anti-cancer compounds are some of the most exciting advances in integrative care…. there exists natural compounds that target the same growth pathways as leading-edge pharmaceuticals (page 505).

Some of natural compounds that have anti-angiogenic properties are berries (most types) which inhibit production of VEGF, a common growth pathway, and also prevent angiogenesis. The soy compound genistein also inhibits VEGF and angiogenesis which may be one reason soy is associated with lower cancer rates. Other natural compounds that can stimulate cells of the immune system to seek out and identify malignant cells are: aloe vera, acemannan, ginseng, curcumin, green tea polyphenols, resveratrol, mushrooms, grape seed extract, etc. (page 505/507)

All the above comments by Dr Block are contained in his bestselling book “LIFE OVER CANCER” which we recommend that you read the whole book or at least chapter 4 “The Anti-Cancer Diet” In this chapter, you will learn why you should not eat the following when you have cancer: –

Dr Block strongly believes that diet affects cancer both directly and indirectly. Nutrients directly impact the mechanisms by which cancer cells grow and spread. They indirectly help control the cancer by changing the surrounding biochemical conditions that either encourage or discourage the progression of malignant disease. The bottom line is that what you eat can spell the difference between conquering your disease or having it rage out of control (page 56).

For more information of the book by Dr Block, visit the following links:

Dr Margaret Cuomo, MD, and a board–certified radiologist wrote the book, “A WORLD WITHOUT CANCER” gave a few tips on “Fighting Cancer with Nutrition and Physical Activity.” Dr Cuomo suggests the following for a Cancer-Prevention Diet: –

a) Eat more fruits and vegetables – such as berries, cruciferous vegetables, tomatoes, dark green, leafy vegetables (page 205).

b) Buy organic – The International Agency for Research on Cancer classifies more than 400 chemicals, including those used in pesticides, as carcinogens (page 206).

c) Eat more Fibre – fibre dilutes the carcinogens in the colon reduce the time in which they remain there, enhanced anti-oxidant action, or produce bacteria that promote, or produce bacteria that promotes a healthy digestive tract (page 206).

d) Avoid Red Meat – a growing body of evidence points to an association between beef, pork, lamb, and goat and cancers of the colon, prostate, pancreas and kidney (page 208/209). Carcinogens may also be present in smoked, salted, or cured meat and in meats cooked at high temperatures.

Besides the above, Dr Cuomo also advise cancer patients to eat more fish, drink green tea, increase consumption of resveratrol, flavor food with turmeric and lastly to limit processed foods (page 207-209).

For further reference, read Dr Cuomo’s article:

Another prominent medical doctor, Dr Russell Blaylock, a board-certified neurosurgeon, believes that “nutrients do block angiogenesis” (pages 182/183)….especially the flavonoids from edible plants such as genistein extracted from soybeans, catechins found in grape-seed extracts, apigenin and luteolin which occur in high concentrations in celery. In his book, “NATURAL STRATEGIES FOR CANCER PATIENTS,” Dr Blaylock advised that doing two things will significantly reduce tumour angiogenesis:

  • Correcting your dietary ratio of omega-6 and omega-3 fats,
  • Increasing your intake of vegetables.

Essentially, it means that a diet of omega-3 products inhibits angiogenesisand a diet high in the omega-6 fats powerfully promotes cancer growth and spread. Nicotine also increases angiogenesis.

A prominent cancer researcher and scientific advisor to the University of Texas Centre for Alternative Medicine, D John Boik, PhD is the author of 2 very scientific texts……CANCER AND NATURAL MEDICINE and NATURAL COMPOUNDS IN CANCER THERAPY. In the 2 books, the subject of angiogenesis is extensively covered.

Some of the natural inhibitors of angiogenesis are curcumin, EPA and DHA, garlic, melatonin, resveratrol, plant flavanoids (genistein, apigenin, luteolin, quercetin, green tea catechins such as EGCG). Read Chapter 8-Natural Inhibitors of Angiogenesis. In this chapter, Dr Boik also pointed out that…”eicosanoids derived from omega-6 fatty acids facilitate cancer progression and eicosanoids derived from omega-3 fatty acids inhibit it.”

Finally, we would like to share with you an E-Book or Nook Book that we found and it is written by Dr Hratch Karamanoukian, MD and a prominent cardiovascular and thoracic surgeon who has specialized in minimally invasive cardiac surgery, thoracic surgery, robotic surgery and vein disorders. In 40 FOODS THAT FIGHT CANCER,” he shares his wisdom as follows:

  • Some foods can help you to fend off cancer, while others could actually be increasing your risk of cancer. Knowing the right foods to add to your diet is very important.
  • Choosing the best foods will be able to help you strengthen and build your immune system, which means fighting off diseases is going to be easier. The right foods are going to make your body stronger and increase your overall health

The following are the 40 Foods that Dr Karamanoukian recommends in his book:

  • Eat more vegetables ……broccoli, cabbage, cauliflower, kale, mushrooms, seaweed, sweet potatoes, turnip greens, onions, summer and winter squash, spinach, olives and Brussels sprouts.
  • Add more fruits to your diet …..tomatoes, avocadoes, grapefruit, figs, oranges, papaya, raspberries, blueberries, strawberries, pears, grapes and lemons.
  • Spices, beans and other foods to help fight cancer …..garlic, sunflower seeds, oregano, turmeric, red wine, peanuts, ginger, tea, brown rice, black beans, ground flaxseed, quinoa, peppermint and fish.




On the main page, click on Foodto view the list of foods profiled as cancer-fighting foods and then click on Evidence for a list of articles to read.


There are a lot more of other such articles…..just google for either anti-angiogenesis or anti-angiogenic foods.

After you have read this far, you would definitely be able to differentiate between foods that inhibit angiogenesis and foods that promote angiogenesis. Remember, your life is in your hands….not in your doctor’s and the choice is yours to decide.

NB: If you are still unsure as to what to cook or how to cook, get hold of a copy of HEALTHY COOKING …A Beginner’s Guide to Preparing Healthy Meals by Ch’ng Beng Im Teo. (ISBN NO: 978-983-2590-25-5).


2. Abnormal Vascular Functions Affect the Tumor EPR Effect

To satisfy the overgrowth of tumor cells, solid tumors need to induce and maintain a dedicated tumor blood supply, which is termed neovascularization. Under inflammatory or hypoxic tumor conditions, cells such as vascular endothelial cells release vascular permeability mediators, resulting in more enhanced tumor vascular permeability than in normal tissue, which can be demonstrated by angiography [25]. However, due to their short half-life and the rapid dilution in the bloodstream, these mediators mainly affect tumor vessels, but not normal tissue blood vessels. In such regions, macromolecules ranging from 10 to 500 nm (e.g., macromolecular anticancer agent, albumin, immunoglobulin, micelles, liposomes, and protein–polymer conjugates) can selectively leak out from the vascular bed and accumulate inside the interstitial space. However, in solid tumors, the EPR effect exhibits great heterogeneity. Tumors show different EPR effects regardless of their types and sizes, patients, or their developmental stages. Tumors with high blood vessel density (e.g., hepatocellular carcinoma) show a strong EPR effect, whereas others with low vascular density (e.g., pancreatic cancer) show a weak EPR effect [5]. Therefore, accurate monitoring and evaluation of the EPR effects in different tumors is essential for the development of personalized EPR-mediated plans for the treatment of tumors.

In principle, due to the widespread presence of EPR in tumors, nanomedicines based on the EPR effect show great promise for improving the efficacy of systemic anticancer drug therapy. However, their full anticancer potential has been hindered because of biological and pathophysiological barriers [26]. Obviously, the vascular system of tumors, which exhibit different vessel density, maturity, perfusion, and pore cutoff size, could be considered one of the main factors that affect the EPR effect [27]. In this review, we summarize the three main approaches through which abnormal tumor blood vessels affect the EPR effect and the related vascular mediators ( Table 1 ).

Table 1

Relationship between tumor vascular-related mediators and three typical vascular characteristics.

FeaturesVascular MediatorsFunctionsTumors with This SubstanceReference
Abnormal angiogenesisVascular endothelial growth factor (VEGF)Key factors in angiogenesis, VEGFs bind to the kinase function of VEGF receptor (VEGFR)-activated receptors, triggering a variety of downstream signaling cascades, such as increased capillary permeability, nitric oxide (NO) production (relaxation of vascular smooth muscle), endothelial cell (EC) proliferation, migration, and survival under stress.Overexpression in most solid tumors[28,29,30]
Tumor necrosis factor (TNF)-αTNF-α mediates monocyte differentiation into angiogenic cells that support tumor angiogenesis. It is also a multipotent proinflammatory cytokine with vascular permeability activity, which can enhance vascular leakage by disrupting the EC adhesion junction VE-cadherin. [22,31,32,33,34,35,36]
Acidic fibroblast growth factor (FGF)/FGF-1Interacts with receptor tyrosine kinase subtypes to induce EC proliferation and maintain tumor angiogenesis. [37]
Basic FGF/FGF-2Controls angiogenesis by inducing the expression of VEGF through paracrine and endocrine mechanisms. [38,39,40]
Platelet-derived growth factor (PDGF)PDGF signals through two cell-surface tyrosine kinase receptors, PDGF receptor α (PDGFRα) and PDGFRβ, and induces angiogenesis by upregulating the production of VEGF and regulating the proliferation and recruitment of perivascular cells. [41,42,43]
Placenta growth factor (PLGF)PLGF only binds to VEGFR-1 and induces tumor angiogenesis, promoting the survival of ECs in tumor-associated blood vessels. [44]
Epidermal growth factor (EGF)A key EGF receptor (EGFR) ligand is one of many growth factors that drive the expression of VEGF. [45]
Hepatocyte growth factor (HGF)Stimulates cell motility and the secretion of proteinases and plays an important role in tumor invasion and progression. [46]
Hypoxia-inducible factor (HIF)-1αUpregulates VEGF gene expression by hypoxia response element binding to the promoter region of VEGF. [47,48,49,50,51]
Transforming growth factor (TGF) -βInduces strong VEGF production in recruited hematopoietic cells, leading to activated angiogenesis and vascular remodeling. Low TGF-β levels contribute to angiogenesis, and high levels of TGF-β can inhibit EC growth. [52,53,54]
Interleukin (IL)-1βInduces angiogenesis indirectly by activating the expression of VEGF in smooth muscle cells. [55]
IL-3Stimulates EC movement and promotes the formation of new blood vessels in vivo. It also stimulates migration and proliferation of vascular smooth muscle cells. [56]
IL-6Regulates the synthesis of VEGF and influences tumor angiogenesis by inducing the production of VEGF. [57]
IL-8Enhances EC survival, proliferation, and matrix metalloproteinase production, and regulates angiogenesis. [58]
Neuropilin 1 and 2Regulates receptor–ligand interactions of the VEGF family. [59]
AdrenomedullinPromotes angiogenesis, protects cells from apoptosis and vascular injury, and affects vascular tone and permeability. [60]
Stromal cell-derived factor 1 (SDF-1), a chemokineSynergizes with VEGF to induce angiogenesis in human ovarian cancer tumors. Furthermore, in invasive breast cancer, stromal fibroblast-derived SDF-1 promotes angiogenesis by recruiting bone marrow-derived endothelial precursors. It plays an angiogenic role through the receptor CXC motif chemokine receptor type 4. [61]
EndostatinInhibits cell cycle control and antiapoptotic genes in proliferating ECs, thus inhibiting angiogenesis. [62]
IntegrinAdhesion molecules such as α6β1 and α6β4 integrins mediate VEGF-induced angiogenesis, which regulates the adhesion of ECs to the ECM, thereby promoting the migration and survival of tumor vasculature. Other integrins (e.g., αvβ3, αvβ5, and α5β1) have also been shown to mediate angiogenesis. [63,64]
Pigment epithelium-derived factor Inhibits angiogenesis via downregulation of VEGF. [65]
Nuclear factor kappa-B (NF-㮫)Activated NF-㮫 can bind to DNA, promote cell proliferation, regulate cell apoptosis, promote angiogenesis, and stimulate invasion and metastasis. [66]
Thyroid hormoneThyroid hormones have proangiogenic effects on ECs and vascular smooth muscle cells initiated by integrin αvβ3 extracellular domain hormone cell-surface receptors. [67]
Matrix metalloproteinases (MMPs)Involved in the process of angiogenesis through its proteolytic role in tissue remodeling, as well as the growth of new blood vessels and the release of angiogenic factors sequestered in the matrix. [68]
Endogenous carbon monoxide (CO) and heme oxygenase (HO)Play an important role in regulating vascular tension and inducing angiogenesis, and can significantly increase vascular permeability and blood flow. [69,70,71,72,73]
AngiogeninUndergoes nuclear translocation in ECs where it stimulates ribosomal RNA transcription and cell proliferation. [74]
Angiopoietin 1Activates matrix-degrading enzymes, including plasminogen activators and MMPs, to loosen the matrix and promote ECs migration. [75]
Vashohibin-1A novel angiogenesis inhibitory protein regulates angiogenesis, inhibits pathological angiogenesis, and promotes tumor vascular maturation by negative feedback.High expression in liver cancer, prostate cancer, renal cancer, and colorectal cancer [76,77]
Vascular permeabilityVEGF
As mentioned above.Overexpression in most solid tumors[28,29,30]
Bradykinin (BK)Activates EC-derived NO synthase, which leads to an increase in NO and plays a role in increasing vascular permeability. [78,79]
Hydroxyprolyl3 BKAs mentioned above.Advanced cancer[80,81,82]
Inducible nitric oxide synthase (iNOS) and NONO is an effective endothelium-derived vascular regulator, which plays an important role in vascular permeability, cell proliferation, and extravasation (EPR effect), inducing vasodilation and increasing blood flow. [83,84,85]
Prostaglandin E1 and I2Usually involved in inflammation and cancer, it has similar effects as NO and can enhance extravasation and EPR effects. [83,86]
TNF-αAs mentioned above. [22,31,32,33,34,35,36]
Angiotensin receptor type 2 (AGTR2)AGTR2 can induce vasoconstriction in healthy tissues and increase systemic blood pressure, and is an effective substance to enhance blood flow and promote vascular permeability in tumors. [87]
IL-2Increased vascular permeability by inducing NO production [76,77]
Endothelin-1 (Et-A, Et-B)Endothelin is an endogenous long-acting vasoconstrictor regulator. [76,77]
Irregular blood flowAGTR2As mentioned above. [87]
Endogenous CO and HOAs mentioned above. [69,70,71,72,73]

2.1. Abnormal Angiogenesis

Angiogenesis is essential for the continuous growth and development of solid tumors. Tumor vessels provide oxygen and nutrients and remove waste products, supply a favorable niche for cancer stem cells, and serve as a conduit for tumor cell metastatic spread and immune cell infiltration. Unlike normal blood vessels, tumor blood vessels with abnormal structure and function impede the delivery of adequate and effective oxygen, as well as therapeutic drugs to cancer cells [88,89]. In cancer progression, the overexpression of proangiogenic factors drives the pathological angiogenesis. An imbalance between local proangiogenic and antiangiogenic factors may lead to the proliferation, migration, and new vessel formation of endothelial cells (EC). Furthermore, pericyte coverage of EC is often absent in the tumor vasculature. Compared with normal tissue with an organized microvasculature with regular branching order, the vascular organization of tumor tissue is disorganized and lacks the conventional hierarchy. Abnormal angiogenesis may lead to structural and functional abnormalities of the vascular system, which are often characterized by tortuous, unorganized, and excessive leakage [90,91]. This feature contributes to the vascular permeability of fluids and the escape of metastatic cancer cells [92,93]. Furthermore, the solid pressure generated by the proliferation of cancer cells compresses the blood and lymphatic vessels in the tumor, further impairing blood and lymphatic flows. These abnormal vascular structures collectively lead to an abnormal tumor microenvironment (TME), characterized by high IFP, hypoxia, and acidosis [88,94,95]. A physiological consequence of these vascular abnormalities is heterogeneity of tumor blood flow, which can interfere with the EPR effect and the uniform distribution of drugs within the tumor.

Tumor cells can promote blood vessel sprouting by releasing angiogenic molecules that bind to their respective receptors in adjacent cells or by paracrine signals [96,97]. Vascular endothelial growth factor (VEGF) appears to play the most critical role in physiological and pathological angiogenesis among all the known angiogenic molecules. It is overexpressed in the majority of solid tumors [28,29] and can promote the survival and proliferation of ECs, increase the display of adhesion molecules on these cells, and increase vascular permeability. By downregulating VEGF signaling in solid tumors, the vasculature may return to a more “normal” state, accompanied by decreased IFP, increased tumor oxygenation, and improved drug permeability in these tumors [98].

In addition to VEGF, other factors and proteins can also promote the abnormal formation of tumor blood vessels. Thus far, 28 proangiogenic factors/genes have been found to mediate tumor angiogenesis [76,77], including the fibroblast growth factor (FGF), hypoxia-inducible factor (HIF), platelet-derived growth factor-B (PDGF-B), tumor necrosis factor-α (TNF-α), chemokines, integrins, and transforming growth factor-β (TGF-β), as well as their receptors [76,99,100,101,102,103]. Acidic and basic FGF (FGF1 and FGF2) have the ability to induce angiogenesis [39]. FGFs stimulate the proliferation and migration of ECs, as well as the production of collagenase and plasminogen activator (PDGF), which stimulate angiogenesis and are related to the aging process of the tumor vasculature in vivo [42,43]. TGF-β possesses dual pro- and antiangiogenic properties. At low levels, TGF-β participates in the switch of angiogenesis by upregulating angiogenic factors and proteinases. At high levels, it can inhibit EC growth, stimulate the differentiation and recruitment of smooth muscle cells, and promote the reorganization of the basement membrane [52]. Moreover, as effective angiogenic factors, chemokines can induce the migration and proliferation of ECs, and they have pro- or antiangiogenic activities [104]. As an angiogenic factor, HIF cooperates with TNF inhibitors to initiate angiogenesis under hypoxic conditions [48,49,50,51]. It activates the signaling pathway and upregulates the expression of VEGF. Growth factors generated by this pathway activate the mitogen-activated protein kinase and protein kinase B signaling pathways, leading to increased levels of HIF-1 protein, thereby promoting tumor angiogenesis. Adhesion molecules (e.g., α6β1 and α6β4 integrins) mediate VEGF-induced angiogenesis, which regulates the adhesion of ECs to the ECM, thereby promoting the migration and survival of tumor vasculature. Other integrins (e.g., αvβ3, αvβ5, and α5β1) also mediate angiogenesis [63,64].

2.2. Irregular Blood Flow

Compared with normal vessels, newly formed tumor vessels are irregular or inconsistent [87]. It has been reported that tumor vessels are insensitive to angiotensin receptor type 2 (AGTR2). In addition, there is intermittent flow (only one flow in 15� min) and reverse flow of blood at the tumor site [105,106]. Moreover, blood often flows in the opposite direction. Irregular blood flow in the tumor is usually caused by irregular vascular structure. Unlike normal tissues, angiogenic factors in tumors at the late stage of vascular maturation will continue to be activated, leading to vascular abnormalities, which are characterized by irregular vascular structure and spatiotemporal heterogeneity [107]. Tumor vessels with irregular structure are characterized by a curved vascular shape, filling of the EC septum, and damage of the basement membrane. These effects lead to distortion of the vascular morphology and high permeability of the vascular EC space [31,108,109,110]. The distortion of blood vessels increases the geometric resistance of blood flow. The high permeability of blood vessels increases the hematocrit of tumor blood, thus increasing the blood viscosity [111]. In addition, the phenomenon of rapid proliferation of tumor cells in a finite space and excessive deposition of ECM can lead to large solid stress between adjacent cells and matrix components. The continuous accumulation of solid stress can lead to the compression of tumor blood vessels and the reduction of cross-sectional area and pressure difference in the direction of blood vessels [112]. The increase in vascular resistance and blood viscosity and the compression of accumulated solid stress significantly increases the resistance to blood perfusion. The increased resistance of tumor vessels to blood perfusion results in a low blood perfusion rate and a slow blood flow rate [113]. The change in blood flow velocity on the transport of nanoparticles through blood vessels has been investigated. A computer simulation explained the effect of blood flow velocity on the transport of nanoparticles. The results showed that the pressure at the vessel wall and the pressure gradient between the vascular wall and interstitial tissue increase in turn with the increase of fluid velocity in the vascular domain. Moreover, the trans-vascular transport efficiency of nanoparticles initially increases and subsequently decreases [114]. In addition, driven by the difference in pressure along the vascular direction, blood perfusion has the characteristics of convection𠄽iffusion. Convection𠄽iffusion differs between tumor blocks and depends on the local pressure gradient and flow resistance due to the heterogeneity of tumor blood vessels [115].

In addition to an irregular structure, the abnormal blood vessels of tumors also exhibit spatiotemporal heterogeneity [116,117]. This heterogeneity indicates the differing distribution of tumor vessels in various parts of the tumor or during the proliferation period. This is mainly indicated by the fact that the distribution of vessels in the periphery of the tumor is usually very rich, while their extension into the interior of the tumor gradually decreases. Therefore, this uneven distribution complicates the delivery of nanodrugs to the tumor center, which seriously hinders the penetration and extravascular transport of such agents. Of note, the high heterogeneity of tumor vessels in experimental mice and humans reduces the antitumor effects of some nanomedicines [26,118].

2.3. Extensive Vascular Permeability

Increased vascular permeability is widely found in endothelium discontinuous tumor vessels such as neovessels and immature vessels, as well as in other pathological tissues with disturbed vascular function. Compared with normal blood vessels, macromolecular drugs can reach the tumor stroma through the leaky vessel wall with large pores without hindrance [12]. However, excessive vascular leakage can cause plasma escape and hemoconcentration. This results in flow stasis and high IFP, which greatly hinder the extravasation of drugs and their movement to the tumor parenchyma. Furthermore, deposited clots of fibrin transiently promote the formation of blood vessels and ECM and prevent the penetration of antitumor therapeutic agents. The vascular media affecting the tumor vascular permeability are summarized below.

Bradykinin (BK) is of great importance in elevating the permeability of inflammatory sites and tumor tissues, thereby maintaining tumor growth [79,81]. Overexpression of BK receptors in solid tumors has been observed, resulting in defective vascular architecture with large intracellular gaps [119]. Kinin can activate EC-derived nitric oxide (NO) synthase, leading to increased levels of NO, a well-established and effective endothelium-derived vascular modulator [85,120,121]. NO is of great significance in vascular permeability, cell proliferation and extravasation (EPR effect), blood vessel dilation, and elevation of blood flow [83,84]. For example, NO generated from l -arginine under the action of NO synthase induces tumor vascular permeability. It has been demonstrated that the inhibition of NO generation can decrease vascular permeability, thereby weakening the EPR effect. This further confirms that NO is inextricably linked to vascular permeability in solid tumors [84,85]. Prostaglandins E1 and I2 are commonly involved in inflammation and cancer, exert similar effects to those of NO, and can enhance extravasation and EPR effects [83,86]. In summary, vascular permeability in tumors is often directly or indirectly related to kinins.

In addition, it has been shown that several vascular mediators, such as vascular permeability factor (VPF), which is important in tumor angiogenesis, TNF-α, and others elevate the vascular permeability of tumors [31]. EC survival and vascular permeability are closely related to the level of VPF/VEGF, as increasing this level can lead to upregulation of the corresponding receptors on ECs. [34,35]. TNF-α, a multifunctional proinflammatory cytokine with vascular permeabilizing effects [22], can enhance vascular leakiness via disrupting the EC adherence junction vascular endothelial cadherin [36]. TNF-α can increase the sensitivity to nanoparticles through serving as a vascular disrupting agent (VDA). At low levels, TNF-α may promote angiogenesis however, at higher concentrations, it destroys the tumor vessels and increases the accumulation of drug in tumors [122].

2 Vasculogenesis

Clear evidence of blood vessel development first appears outside of the embryo proper, on the yolk sac, as focal aggregations of mesenchymal cells, known as blood islands, form within the mesoderm adjacent to the extraembyronic endoderm. While the earliest studies suggested that the embryo proper becomes vascularized from branching and growth of extraembyronic blood vessels which in turn are derived from fusion and channeling of blood islands, intraembryonic origins of a vascular network were recognized as early as 1900 when it was determined that cells from isolated embryos were able to give rise to blood vessels. It was at this time that the term angioblast, referring to the cells from which all endothelial cells arise, was described. It was also during this early period that the presence of the hemangioblast — a common precursor for both endothelial cell and hematopoietic cell — was first postulated ( Fig. 2). Notably, it was almost 100 years later that the presence of the hemangioblast was confirmed [5]. Differentiation of pluripotent embryonic precursor cells into hemangioblastic cells is induced to at least some extent by fibroblast growth factor (FGF) via protein kinase C signaling [6]. Hemangioblasts undergo their first critical steps of differentiation within the blood islands. Cells at the perimeter of the blood islands give rise to precursors for endothelial cells, while those at the center constitute hematopoietic precursors. The molecular signals determining the fate of the hemangioblast are not fully elucidated. However, several genes have been identified that may play a role in this early event [7]. These include Ets-1 [8], Hex [9], Vezf [10], Hox [11,12], members of the GATA-family, basic helix-loop-helix (bHLH) factors [13,14], and the Id-proteins [15]. The earliest markers common to endothelial and hematopoietic precursors so far identified are CD31, CD34 and the receptor tyrosine kinase type-2 of vascular endothelial cell growth factor (VEGFR-2 or KDR/Flk1) [16]. Inactivation of the VEGFR-2 gene in mice results in embryonic lethality, with lack of development of both hematopoietic and endothelial cell lineages, supporting the critical importance of this receptor at that developmental stage [17], although not defining the steps regulating differentiation into endothelial versus hematopoietic cell.

A common origin for the two types of blood-vessel cell. Endothelial and smooth muscle cells arise from separate types of precursor. Endothelial cells arise from precursors called angioblasts or hemangioblasts in the embryo, or from circulating endothelial progenitors in the adult. Angioblasts give rise to arterial and venous lineages. Smooth muscle cells and pericytes, by contrast, can form from a variety of progenitors. These include mesenchymal cells, neural crest cells, and progenitors in the epicardium in the embryo. Progenitors in the bone marrow and its stroma, and mesenchymal myofibroblasts also give rise to smooth muscle cells. A new common vascular progenitor cell that gives rise to both types of blood-vessel cell has been recently identified. Vascular endothelial growth factor (VEGF) promotes the development of endothelial cells from this precursor. TGF-β1 has been involved in differentiation of mesenchymal cells to progenitors, that express the receptor for PDGF-BB. The latter stimulates their development into smooth muscle cells and pericytes and is responsible for their recruitment around nascent vessels. Adapted from Ref. [114].

A common origin for the two types of blood-vessel cell. Endothelial and smooth muscle cells arise from separate types of precursor. Endothelial cells arise from precursors called angioblasts or hemangioblasts in the embryo, or from circulating endothelial progenitors in the adult. Angioblasts give rise to arterial and venous lineages. Smooth muscle cells and pericytes, by contrast, can form from a variety of progenitors. These include mesenchymal cells, neural crest cells, and progenitors in the epicardium in the embryo. Progenitors in the bone marrow and its stroma, and mesenchymal myofibroblasts also give rise to smooth muscle cells. A new common vascular progenitor cell that gives rise to both types of blood-vessel cell has been recently identified. Vascular endothelial growth factor (VEGF) promotes the development of endothelial cells from this precursor. TGF-β1 has been involved in differentiation of mesenchymal cells to progenitors, that express the receptor for PDGF-BB. The latter stimulates their development into smooth muscle cells and pericytes and is responsible for their recruitment around nascent vessels. Adapted from Ref. [114].

As the yolk sac vasculature begins to form ∼7.5 days post coitum (dpc) in the mouse, angioblasts that have migrated to the paraxial mesoderm assemble into aggregates, proliferate and subsequently differentiate to form a plexus with endocardial tubes, leading to formation of the dorsal aortae, cardinal veins and the embryonic stems of yolk sac arteries and veins ( Fig. 3). Vasculogenesis was originally believed to be restricted to embryonic development. However, it has been established that angioblasts not only migrate intraembryonically, but may circulate post-natally, and may be recruited for in situ vessel growth [18–20] ( Fig. 4). Unlike shed cells, circulating CD34/VEGFR-2/AC133-positive marrow-derived angioblasts in the adult have a high proliferation rate [21]. How angioblasts ‘know’ where and when to initiate vasculogenesis is largely a mystery, although a variety of growth factors and receptors, including vascular endothelial growth factor (VEGF), granulocyte monocyte-colony stimulating factor (GM-CSF) and other cytokines have been implicated, as they have been shown to be able to recruit bone-marrow derived angioblasts to sites of neovascularization postnatally [18,22,23]. Angioblast differentiation may be promoted by VEGF, VEGFR2 and basic fibroblast growth factor (bFGF) [24–27], while VEGF receptor-1 (VEGFR-1 Flt1) has been determined to suppress hemangioblast commitment [28]. The findings that endothelial cells still develop in mice engineered to lack VEGF but do not develop any longer in the absence of VEGFR-2, suggests that there are additional molecules, binding to VEGFR-2, that determine endothelial cell fate. Although vasculogenesis is closely linked to hematopoiesis, precursors of definitive hematopoietic cells arise only from the dorsal aorta, and subsequently populate the liver, spleen and bone marrow. A basic understanding of the molecular mechanisms that separate hematopoiesis from vasculogenesis will not only impact on angiogenic therapies, but also on treatment of a variety of hematologic disorders.

Pathological vascular growth in the adult may occur via vasculogenesis (angioblast mobilization), angiogenesis (sprouting) or arteriogenesis (collateral growth). With permission from Ref. [4].

Pathological vascular growth in the adult may occur via vasculogenesis (angioblast mobilization), angiogenesis (sprouting) or arteriogenesis (collateral growth). With permission from Ref. [4].

VEGF initiates assembly of endothelial cells (EC), PDGF-BB recruits pericytes (PC) and smooth muscle cells (SMC), whereas angiopoietin-1 (Ang1) and TGF-β1 stabilize the nascent vessel. Angiopoietin-2 (Ang2) destabilizes the vessel, resulting in angiogenesis in the presence of angiogenic stimuli, or in vessel regression in the absence of endothelial survival factors. With permission from Ref. [4].

VEGF initiates assembly of endothelial cells (EC), PDGF-BB recruits pericytes (PC) and smooth muscle cells (SMC), whereas angiopoietin-1 (Ang1) and TGF-β1 stabilize the nascent vessel. Angiopoietin-2 (Ang2) destabilizes the vessel, resulting in angiogenesis in the presence of angiogenic stimuli, or in vessel regression in the absence of endothelial survival factors. With permission from Ref. [4].

Blood Clotting: Mechanisms and Stages | Blood | Hematology | Biology

In this article we will discuss about the mechanisms and stages of blood clotting.

Mechanism of Blood Clotting:

Blood Clotting is one of three mechanisms that reduce the loss of blood from broken blood vessels.

The three Mechanisms are:

The smooth muscle in blood vessel walls contracts immediately the blood vessel is broken. This response reduces blood loss for some time, while the other haemostatic mechanisms become active.

ii. Platelet Plug Formation:

When blood platelets encounter a damaged blood vessel they form a “platelet plug” to help to close the gap in the broken blood vessel. (The key stages of this process are called platelet adhesion, platelet release reaction, and platelet aggregation)

Following damage to a blood vessel, vascular spasm occurs to reduce blood loss while other mechanisms also take effect. Blood platelets congregate at the site of damage and amass to form a platelet plug. This is the beginning of the process of the blood “breaking down” from its usual liquid form in such a way that its constituents play their own parts in processes to minimize blood loss.

Blood normally remains in its liquid state while it is within the blood vessels but when it leaves them the blood may thicken and form a gel (coagulation). Blood clotting (technically “blood coagulation”) is the process by which (liquid) blood is transformed into a solid state.

This blood clotting is a complex process involving many clotting factors (incl. calcium ions, enzymes, platelets, damaged tissues) activating each other.

Stages of Blood Clotting:

1. Formation of Prothrombinase:

Prothrombinase can be formed in two ways, depending of which of two “systems” or “pathways” apply.

This is initiated by liquid blood making contact with a foreign surface, i. e. something that is not part of the body or

This is initiated by liquid blood making contact with damage tissue.

Both the intrinsic and the extrinsic systems involve interactions between coagulation factors. These coagulation factors have individual names but are often referred to by a standardised set of Roman Numerals, e.g. Factor VIII (anti-haemophilic factor), Factor IX (Christmas factor).

2. Prothrombin Converted Into the Enzyme Thrombin:

Prothrombinase (formed in stage 1.) converts prothrombin, which is a plasma protein that is formed in the liver, into the enzyme thrombin.

3. Fibrinogen (Soluble) Converted to Fibrin (Insoluble):

In turn, thrombin converts fibrinogen (which is also a plasma protein synthesized in the liver) into fibrin.

Fibrin is insoluble and forms the threads that bind the clot

There are two pathways that lead to the conversion of prothrombin to thrombin:

(1) The intrinsic pathway and

(1) Intrinsic Pathway:

The intrinsic pathway, which is triggered by elements that lie within the blood inself (intrinsic to the blood), occurs in the flowing way. Damage to the vessel wall stimulates the activation of a cascade of clotting factors (for the sake of simplicity we will not consider the individual factors). This cascade results in the activation of factor X.

Activated factor X is an enzyme that converts prothrombin to thrombin. Thrombin converts fibrinogen to fibrin monomers, which then polymerize in fibrin fibers. Fibirin fibers form a losse meshwork that is stabilized by crosslinks created by factor XIII. The stabilized meshwork of fibrin fibers ins now a clot that traps red blood cells and platelets and thus stops the flow of blood.

(2) Extrinsic Pathway:

The extrinsic pathway is triggered by tissue damage outside of the blood vessel. This pathway acts to clot blood that has escaped from the vessel into the tissues. Damage to tissue stimulates the activation of tissue thromboplastin, an enzyme that catalyzed the activation of factor X. At this point the intrinsic and extrinsic pathways converge and the subsequent steps are the same as those described above.

With advanced atherosclerosis take one baby asprin yet day to reduce the probability of heart attack and stroke.

Small tears of the capillaries and arterioles are happening all the time Platelets are responsible for quickly sealing these tears before the slower process of clotting completes the job.

In the absence of adequate numbers of platelets these micro blotches (thrombocytopenia purpura) visible on the skin. Thrombocytopenia can be acute or chronic and has many causes. Severe, untreated cases result in death.

The blood contains about a dozen clotting factors. These factors are proteins that exist in the blood in an inactive state, but can be called into action when tissues or blood vessels are damaged.

The activation of clotting factors occurs in a sequen­tial manner. The first factor in the sequence activates the second factor, which activates the third factors and so on. This series of reactions is called the clotting cascade.

Blood clotting is the transformation of liquid blood into a semisolid gel. Clots are made from fibers (polymers) of a protein called fibrin. Fibrin monomers come from an inactive precursor called fibrinogen.

The body of the fibrinogen molecule has caps on its ends that mast fibrin-to-fibrin binding sites. If the caps are removed then fibrin monomers polymerize to form fibrin polymers. This process required thrombin the enzyme that converts fibrinogen to fibrin.

This process also requires calcium, which acts as a kind of glue to hold the fibrin monomers to each other to form the polymeric fiber. The fibrin fibers form a loose mesh work that is stabilized by clotting factor XIII. The stabilized meshwork of fibrin fibers traps erythrocytes, thus forming a clot that stops the flow of blood.

Clot Busting Drugs:

Blood clots can be life-threatening if they form inappropriately in critically locations. Clots that block coronary arteries cause the heart attacks, while clots that block arteries in the brain cause stroke. Drugs that can mediate the removal of clots, “clot busters”, are used in cases of heart attract and stroke to decrease the damage caused by the clot.

Drugs used clinically to remove cots include:

1. Tissue plasminogen activator (TPA) was recently cloned and is now produced in mass quantities by the biotech fig, Amgen. It is used clinically to dissolved clots in coronary arteries following a heart attack. It is also used to dissolved clots in the brain following stroke.

2. Streptokinase is an enzyme that directly dissolved blood clots. It is produced by streptococcus bacteria. The bacteria use streptokinase to dissolve clots that nega­tively affect their growth in the human host. This clot dissolving enzyme is appar­ently as effective as recombinant TPA.

Streptokinase cost $2 dollars per does while TPA costs $2000 dollars per dose. Based on economic concerns, streptokinase is the drug of choice. However, streptokinase is not a human enzyme, therefore the immune system sees it as a foreign molecule that should be distorted.

The immune response increases with repeated use of this limits the effectiveness of the drug over time. TPA, on-the-other-hand is a huna molecule whole which the im­mune system does not destroy.

Angiogenesis and Cancer

Angiogenesis refers to the formation of new blood vessels due to the development of existing capillaries and postcapillary veins. Accurately speaking, angiogenesis is not the same as vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors, and neovascularization. The first vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most, if not all, blood vessel growth during development and in disease.

Angiogenesis is a normal and vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, it is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Tumor angiogenesis is an extremely complex process that generally includes steps such as degradation of the vascular endothelial matrix, migration of endothelial cells, proliferation of endothelial cells, formation of vascular loops by the branching of endothelial cells, and the formation of a new basement membrane. Because the neovascular structure and function of the tumor tissue are abnormal, and the vascular matrix is incomplete, the microvessels are prone to leakage. Therefore, the tumor cells do not need to go through a complicated invasion process and penetrate directly into the bloodstream and metastasis. A growing number of studies have shown that benign tumors have sparse angiogenesis and slow blood vessel growth, while most malignant tumors have intensive angiogenesis and rapid growth. Therefore, angiogenesis plays an important part in the development and metastasis of tumors, and it is a marker for most malignant tumors. Inhibiting this process will significantly prevent the development and spread of tumor tissue.

Regulation Mechanisms

Angiogenesis is performed by various angiogenic proteins e.g integrins and prostaglandins, including several growth factors, such as VEGF, 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. In general, FGFs stimulate a variety of cellular functions by binding to cell surface FGF-receptors in the presence of heparin proteoglycans. The FGF-receptor family has seven members, and all the receptor proteins are single-chain receptor tyrosine kinases that become activated through autophosphorylation induced by a mechanism of FGF-mediated receptor dimerization. Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses, including cell differentiation, proliferation, and matrix dissolution, thus initiating a process of mitogenic activity critical for the growth of endothelial cells, fibroblasts, and smooth muscle cells. FGF-1 can bind to all seven FGF-receptor subtypes, making it the broadest-acting member of the FGF family, and a potent mitogen for the diverse cell types needed to mount an angiogenic response in damaged tissues, where upregulation of FGF-receptors occurs. 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.

Besides FGF-1, 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. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are critical players in wound-healing. They stimulate the proliferation of fibroblasts and endothelial cells that give rise to angiogenesis and developing granulation tissue both increase blood supply and fill up a wound space early in the wound healing process.

Vascular endothelial growth factor (VEGF), a homodimer glycoprotein encoded by a single gene, can directly stimulate the migration, proliferation and division of vascular endothelial cells, and increase microvascular permeability. It is aimed at the highest specificity of endothelial cells and the strongest mitogenic effect of mitogens. In vitro studies clearly demonstrate that VEGF is a potent stimulator of angiogenesis because, in the presence of this growth factor, plated endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries. VEGF causes a massive signaling cascade in endothelial cells. Binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producing NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. In this process, VEGF binds with high affinity to two receptors KDR and Flt-1 on endothelial cells, directly stimulates the proliferation of vascular endothelial cells, and induces their migration and formation of lumen-like structures at the same time, it also increases microvascular permeability and induces plasma proteins (mainly fibrinogen) is extravasated and promotes neovascularization in vivo by inducing interstitial production. VEGF plays a central regulatory role in angiogenesis and formation and is a key angiogenesis stimulator.

The angiogenesis process requires interactions between vascular endothelial cells (EC) and the extracellular matrix, between EC and EC, and between EC and other surrounding cells. This role is accomplished by adhesion factors, in which matrix metalloproteinase (MMP) initiates the activation and migration of endothelial cells by degrading the basement membrane glycoprotein and extracellular matrix components, and the integrin family mediated by binding to different ligands. The migration and adhesion of vascular endothelial cells contribute to the maturation and stability of neovascularization, and cell adhesion factor (ICAM-1) can produce immunosuppression and reduce the cytotoxicity of natural killer cells, helping ectopic tissue to escape the immune system of the body. The killing of natural killer cells promotes angiogenesis after invading ectopic tissue.

The angiogenesis mechanism is complex, and many factors involved in and promote angiogenesis. The number of macrophages in the peritoneal fluid of epithelial mesenchymal transitions (EMT) is significantly increased. Macrophages secrete TNF-α and IL-8, and then they can promote the proliferation of vascular endothelial cells. Besides, transforming growth factor-β ( TGF-β), platelet-derived endothelial cell growth factor (PD-ECGF), heparanase, angiogenin (angs), osteogenin (OPN), cyclooxygenase (COX-2), hypoxia-inducible factor -1, Laminin (LN), placenta growth factor (PLGF), Survivin, erythropoietin (Epo) are involved in the formation of EMT blood vessels.

Endostatin (ENS) is a C-terminal fragment of XVIII collagen that specifically inhibits endothelial cell proliferation and promotes apoptosis. It inhibits angiogenic factors such as VEGF and bFGF and their biological effects, and can also interact with MMPs and integrin ανβ3, Ανβ5. In addition, ENS can inhibit the migration and adhesion of endothelial cells and macrophages. It has a strong ability to inhibit neovascularization, and plays an important role in the regulation of tumor angiogenesis.

In angiogenesis, there is a substance that inhibits the growth of new blood vessels, is called angiogenesis inhibitor. They include: Angiostain, it can selectively inhibit endothelial cell proliferation Thrombospondin-1 (TSP-1), it inhibits angiogenesis induced by VEGF or bFGF by interacting with the cell matrix and is concentration-dependent Tissue inhibitors of metalloproteinases (TIMPs), it can inhibit angiogenesis through the formation of complexes with MMPs, thereby inhibiting angiogenesis. In addition, platelet factor-4 (PF-4), interferon-α (IFN-α), interleukin-13, interleukin-4, interleukin-10, and plasminogen activator inhibitor all inhibit the process of blood vessel formation.

Clinical Significance

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A malignant tumor consists of a population of rapidly dividing and growing cancer cells that progressively accrues mutations. However, tumors need a dedicated blood supply to provide the oxygen and other essential nutrients they require in order to grow beyond a certain size. So angiogenesis is essential for tumor growth and metastasis, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer progression. Tumor angiogenesis is an extremely complex process that generally includes steps such as degradation of the vascular endothelial matrix, migration of endothelial cells, proliferation of endothelial cells, formation of vascular loops by the branching of endothelial cells, and the formation of a new basement membrane. Tumor angiogenesis occurrence depends on an interaction effect. On the one hand, tumor cells release angiogenic factors to activate vascular endothelial cells and promote endothelial cell proliferation and migration. On the other hand, endothelial cells also secrete certain angiogenic growth factors to stimulate the growth of tumor cells. The interaction between tumor cells and endothelial cells runs through the whole process of tumor angiogenesis from beginning to end. In general, tumor-derived capillaries are formed by extending and expanding on the basis of the original blood vessels, and the process is similar to the typical process of wound healing and embryogenesis. These neovascularizations provide nutrients for the continuous infiltration of the growing primary tumor. In turn, the tumor cells secrete a variety of substances during growth to accelerate the formation of tumor-derived capillaries.

Similar with the common angiogenesis, FGF, VEGF and other angiogenic stimulator is also necessity for the associated-tumor angiogenesis. For instance, VEGF can promote endothelial cell proliferation, increase vascular permeability, and promote the expression of plasminogen activator(PA) and plasminogen activator inhibitor (PAI), interstitial collagenase and thrombin in endothelial cells, and extravasation of plasma fibrin, leading to deposition of cellulose in tumor stroma and promotion of macrophage Cells, fibroblasts, and endothelial cells grow, leading to tumor angiogenesis and play an important role in tumor growth. Most of the VEGF secreted by tumor cells is concentrated around tumor blood vessels. The response of tumor blood vessels to VEGF was higher than that of normal blood vessels, indicating that VEGF is closely related to tumor angiogenesis. In cell transfection experiments, Me157 melanoma cells transfected with VEGF gene can secrete a large amount of VEGF. After inoculated subcutaneously in nude mice, a large number of blood vessels appear in the tumor tissue, and they pass radially through the tumor by radial, suggesting that it not only affects the number of angiogenesis of the tumor, but also affects the structure of angiogenesis. Therefore, clinical trials with tumor angiogenesis as a target have attracted much attention. Finding a way that can cure the tumor by inhibiting tumor angiogenesis is a hot research direction in cancer therapy.

Targeted cancer drug combined with low-dose chemotherapy shrinks tumors, slows ovarian cancer

The study, presented at the 41st annual meeting of the American Society of Clinical Oncology (ASCO) in Orlando, Florida, evaluated 29 patients with recurrent ovarian cancer after having undergone up to three rounds of treatment with standard chemotherapy. All patients received a low dose of chemotherapy daily (taken in pill form) and bevacizumab by intravenous infusion. The investigators found that nearly half of the patients had no progression of their ovarian cancer six months after receiving treatment with bevacizumab and low-dose oral chemotherapy. In addition, tumors shrank in over 20 percent of patients.

"Our early results suggest that this targeted drug worked effectively with a pill form of low-dose chemotherapy to shrink or stop the growth of ovarian cancer in patients whose disease had recurred after prior treatment with standard chemotherapy," Garcia said.

Bevacizumab is the common name for Avastin TM , a monoclonal antibody that targets and stalls the function of a substance made by cells called the vascular endothelial growth factor (VEGF), which stimulates the growth of blood vessels that nourish cancer tumors and cause them to grow - a process called angiogenesis. Currently, bevacizumab has been approved by the Food and Drug Administration as a first-line treatment in combination with 5-FU-based chemotherapy for patients with colon cancer that has spread to other parts of the body.

Cyclophosphamide is a standard chemotherapy drug given intravenously or as a pill that is used to treat several types of cancer often in combination with other drugs. But laboratory researchers have found that when the drug is given at a lower dose over a prolonged period, the dosage is too low to kill cancer cells, but can stop blood vessel growth that feeds the tumor.

"Our theory was that if we could combine a known anti-angiogenesis agent with a lower dose of chemotherapy on a prolonged basis, the two would work synergistically to cut off the blood supply feeding the ovarian cancer tumor and stop the cancer from growing," Garcia said.

To find out whether bevacizumab and cyclophosphamide would shrink tumors and increase the survival of patients with recurrent ovarian cancer, the investigators evaluated 29 patients whose disease had recurred after at least one and up to three prior rounds of treatment with standard chemotherapy. Patients received 50 milligrams of a cyclophosphamide pill daily and 10 milligrams per kilogram of bevacizumab intravenously once weekly for the first three weeks of the study and every two weeks thereafter.

The investigators found that 47 percent of patients' had no progression of their disease at six months of treatment with both bevacizumab and cyclophosphamide. Further, ovarian cancer tumors shrank in 21 percent of the patients, while 59 percent achieved stable disease - or ovarian cancer that did not progress or diminish for at least two months of treatment. Side effects were similar to those reported in other studies of bevacizumab, including high blood pressure, fatigue, and blood clots.

"Our study suggests that an anti-angiogenesis cancer drug used in combination with a low dose of chemotherapy - which was conveniently taken in pill-form - shrank tumors and may delay progression of the disease in a significant number of patients with recurrent ovarian cancer," Garcia said. "These early results may lead to a new use of this monoclonal antibody in the treatment of ovarian cancer."

Ovarian cancer is the seventh most common cancer among women and ranks fourth as the cause of cancer death in women. The American Cancer Society estimates that about 22,220 women in the United States will develop ovarian cancer this year and that about 16,210 women will die from the disease. Typically, ovarian cancer is treated with surgery, chemotherapy and radiation, depending on how far the cancer has spread. To date, no standardized screening test is available to detect ovarian cancer.

This study was sponsored by the National Cancer Institute under its Cooperative Research and Development Agreement with Genentech for the development of bevacizumab. It was performed as part of the California Cancer Consortium, a collaborative National Cancer Institute (NCI) funded group consisting of Cedars-Sinai Medical Center Princess Margaret Hospital University of Chicago University of California, Davis City of Hope and University of Southern California Norris Cancer Center.

One of only five hospitals in California whose nurses have been honored with the prestigious Magnet designation, Cedars-Sinai Medical Center is one of the largest non-profit academic medical centers in the Western United States. For 17 consecutive years, it has been named Los Angeles' most preferred hospital for all health needs in an independent survey of area residents. Cedars-Sinai is internationally renowned for its diagnostic and treatment capabilities and its broad spectrum of programs and services, as well as breakthroughs in biomedical research and superlative medical education. It ranks among the top 10 non-university hospitals in the nation for its research activities and was recently fully accredited by the Association for the Accreditation of Human Research Protection Programs, Inc. (AAHRPP). Additional information is available at

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Materials and Methods

Chemicals and cell lines.

SU11248, Sunitinib, was purchased from LC Laboratories (Woburn, MA). Human VEGF protein was obtained from PROSPECT-TANY TECHNOGENE LTD., (Israel). The hormone, 17-beta-estradiol was purchased from Sigma (St. Louis, MO). The mouse breast cancer cells (E0771), which were originally isolated from an immunocompetant C57BL/6 mouse, were provided by Dr. Sirotnak FM at Memorial Sloan Kettering Cancer Center, New York, NY. 7 Human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HASMC), human estrogen-receptor positive breast cancer (MCF-7) cells and human estrogen-receptor negative breast cancer (MDA-MB-231) cells were purchased from the American Type Culture Collection (Rockville, MD).

Animal protocols.

The protocols were carried out according to the guidelines for the care and use of laboratory animals implemented by the National Institutes of Health and the Guidelines of the Animal Welfare Act, and were approved by the University of Mississippi Medical Center's Institutional Animal Care and Use Committee. Seven week-old female C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, Maine). The mice were allowed to acclimate for 1 week with standard chow diet (Teklad, Harlan Sprague Dawley Indianapolis, IN) and tap water before beginning the experiment. The eight week old female mice (n = 8) were given SU11248 10 mg/50 ml in drinking (distilled) water for 4 weeks, and the control group (n = 8) was given regular drinking (distilled) water only. Each mouse (20 g) drank 2 to 4 ml of water per day. Therefore, the mice consumed 20 to 40 mg/kg/day of SU11248. During the 2 nd week, all mice were inoculated subcutaneously on the left pad of the fourth mammary gland with 100 µl of 10 6 E0771 cells suspended in phosphate-buffered saline, using a 23-gauge needle. The body weight of the mice was monitored weekly. Tumor size was monitored every other day in two perpendicular dimensions parallel with the surface of the mice using dial calipers. At the end of the experiment, the tumors were removed and weighted for analysis. Then, they were placed into either liquid nitrogen for total protein extraction and nuclear protein extraction or 10% neutral formalin for immunohistological study.

Morphometric analysis of tumor angiogenesis.

The quantification of blood vessels in tumor tissues was determined by our previously reported methods. 6 , 9 Consecutive thin cryosections (5 µm) of OCT compound (Sakura Finetek, Torrance, CA) embedded tumor tissues were fixed in acetone at 4ଌ for 10 min. After washing in phosphate-buffered saline (PBS), the sections were treated with 3% H2O2 for 10 minutes to block endogenous peroxidase activity, and then blocked with normal rabbit serum. Then, the sections were washed in PBS and incubated with rat anti-mouse CD31 (PECAM-1) monoclonal antibody (BD Pharmingen, San Diego, CA) at a 1:200 dilution overnight at 4ଌ. Negative controls were incubated with rat serum IgG at the same dilution. All sections were washed in PBS containing 0.05% Tween-20, and then incubated with a 2 nd antibody, mouse anti-rat IgG (Vector laboratories, Burlingame, CA), at a 1:200 dilution for 1 hour at room temperature, again followed by washing with PBS containing 0.05% Tween-20. The sections were incubated in a 1:400 dilution of Extravadin Peroxidase (Sigma, St. Louis, MO) for 30 min. After washing in PBS containing 0.05% Tween-20, the sections were incubated in peroxidase substrate (Vector laboratories, Burlingame, CA) for 5 min. The sections were washed in PBS containing 0.05% Tween-20 and were counterstained with hematoxylin. A positive reaction was indicated by brown staining. The microvascular vessels were quantified by manual counting under light microscopy. A microscopic field (0.7884 mm 2 ) was defined by a grid laced in the eye-piece. At least 20 microscopic fields were randomly taken from each tumor for analysis. Any endothelial cell or cell cluster showing antibody staining and clearly separated from an adjacent cluster was considered to be a single, countable microvessel. The value of average microvascular density (AMVD) was determined by calculating the mean of the vascular counts per mm 2 obtained in the microscopic fields for each tumor sample.

Reverse transcription polymerase chain reaction.

The total RNA isolation from cultured E0771 cells was performed as previously described. 9 The VEGFR1 mRNA and VEGFR2 mRNA of E0771 cells were determined by RT-PCR as previously described. 10 Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed with an iScript cDNA synthesis kit from Bio-Rad following the manufacturer's instructions: 2 µg total RNA, 4 µl 5X iScript reaction mix, 1 µl iScript reverse transcriptase in a total volume of 20 µl. The RT mixture was incubated at 25ଌ for 5 min, 42ଌ for 30 min, and 4ଌ for 5 min in a programmable thermal cycler (Bio-Rad). The PCR mixture included 5 µl of the RT mixture, 2 µl sense primer, 2 µl antisense primer and 25 µl Taq mix in a total volume of 50 µl. The amplifications were performed as follows: 40 cycles at 94ଌ for 5 min/94ଌ, 0.5 min 60ଌ for 0.5 min and 72ଌ for 1 min/72ଌ, 7 min, then 4ଌ. The following primer sequences were used: VEGFR1 sense 5′-GAG GAG GAT GAG GGT GTC TAT AGG T-3′ and antisense 5′-GTG ATC AGC TCC AGG TTT GAC TT-3′ (115 bp) VEGFR2 sense 5′-GCC CTG CTG TGG TCT CAC TAC-3′ and antisense 5′-CAA AGC ATT GCC CAT TCG AT-3′ (115 bp). The VEGFR1 mRNA and VEGFR2 mRNA were observed by running the RT-PCR products on 2% agarose gel with TAE buffer.

Proliferation assay.

E0771 cells, MCF-7 cells and MDA-MB-231 cells were maintained as monolayer cultures in RPMI Medium 1640 (GIBCO) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B and incubated at 37ଌ in a humidified 5% CO2/air injected atmosphere. The cell lines of human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) were cultured using M199 media (GIBCO) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B and incubated at 37ଌ in a humidified 5% CO2/air injected atmosphere. When the monolayer reached approximately 80% confluence, the cells were washed with PBS and incubated with fresh media with 10% FBS in the absence (vehicle control) and presence of VEGF (10 ng/ml), VEGF plus SU11248 (10 µmol/L) or SU11248 (5 and 10 µmol/L) for 18 hrs. 3H-thymidine incorporation assay was used to determine the cell proliferation during the last 6 hours of incubation as previously described. 11

Migration assay.

Migration was determined using BD BioCoat Matrigel Invasion Chamber (BD Bioscience Discovery Labware, Sedford, MA) as described in a previous study, in which only invasive cells digested the matrix and moved through the insert membrane. 12 1 × 10 5 E0771 cells per well in 0.5 ml medium (RPMI Medium 1640) were seeded in the matrigel-coated upper compartment (insert) of a Transwell (24-well format, 8 µm pore) in the absence of SU11248 (control) and presence of SU11248 (10 µmol/L) and the medium with 10% FBS was added to the lower part of the well. After overnight incubation at 37ଌ and 5% CO2, cells on the upper surface of the insert were removed using a cotton wool swab. Migrated cells on the lower surface of the insert were stained using DiffQuit (Dada Behring, D࿍inen, Switzerland). The images of migrated cells were taken and the number of migrated cells was counted using a microscope (Leica, Germany) in a 20x objective.

Measurements of protein levels of VEGF and VEGFR1 by ELISA.

Protein levels of VEGF and VEGF receptor-1 in cultured MCF-7 and MDA-MB-231 cells were determined using mouse VEGF and VEGF Flt-1 ELISA kits (Rɭ Systems, Minneapolis, MN), according to the manufacturer's instructions. The total proteins of cultured MCF-7 or MDA-MB-231 cells were extracted using NE-PER Cytoplasmic Extraction Reagents (Pierce, Rockford, IL), according to the manufacturer's protocol. Protein levels of VEGF and VEGFR1 (Flt-1) in cultured MCF-7 and MDA-MB-231 cells were determined in the absence (vehicle control) and presence of 17beta-estradiol (5 nmol/L) for 18 hrs. The total protein concentration of cultured MCF-7 and MDA-MB-231 cells was determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein concentrations of VEGF and VEGFR1 were normalized and expressed as pictograms per milligram of total cellular protein.

Statistical analyses.

All determinations were performed in duplicated sets. Where indicated, data is presented as mean ± SE. Statistically significant differences in mean values between the two groups were tested by an unpaired Student's t-test. ANOVA was used to analyze the differences between two groups with multiple comparisons. A value of p < 0.05 was considered statistically significant. All statistical calculations were performed using SPSS software (SPSS Inc., Chicago, IL).