Angiogenesis

Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over this, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for new blood vessel formation by splitting off existing ones.

Angiogenesis is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state.

Additional recommended knowledge

What is the Correct Way to Check Repeatability in Balances?

Safe Weighing Range Ensures Accurate Results

Essential Laboratory Skills Guide

Types of angiogenesis

Sprouting angiogenesis

Sprouting angiogenesis was the first identified form of angiogenesis. It occurs in several well-characterized stages. First, biological signals known as angiogenic growth factors activate receptors present on endothelial cells present in pre-existing venular blood vessels. Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basement membrane in order to allow endothelial cells to escape from the original (parent) vessel walls. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelial cells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, called integrins. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels ^[1].

Intussusceptive angiogenesis

Intussusception, also known as splitting angiogenesis, was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis. First, the two opposing capillary walls establish a zone of contact. Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen. Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen. Finally, the core is fleshed out with no alterations to the basic structure. Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.

Modern Terminology of Angiogenesis

Besides the differentiation between “Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists the today more common differentiation between the following types of angiogenesis:

Vasculogenesis – Formation of vascular structures from circulating or tissue-resident endothelial stem cells (angioblasts), which proliferate into de-novo endothelial cells. This form particularly relates to the embryonal development of the vascular system.

Angiogenesis – Formation of thin-walled endothelium-lined structures with/without muscular smooth muscle wall and pericytes (fibrocytes). This form plays an important role during the adult life span, also as "repair mechanism" of damaged tissues.

Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.

Because it turned out that even this differentiation is not a sharp one, today quite often the term “Angiogenesis” is used summarizing all different types and modifications of arterial vessel growth.

References

• Rubanyi, G.M. (Ed): Angiogenesis in health and disease. M.Dekker, Inc., New York – Basel, 2000
• Raizada, M.K., Paton, J.F.R., Kasparov, S., Katovich, M.J. (Eds): Cardiovascular genomics. Humana Press, Totowa, N.J., 2005
• Kornowski, R., Epstein, S.E., Leon, M.B.(Eds.): Handbook of myocardial revascularization and angiogenesis. Martin Dunitz Ltd., London, 2000
• Stegmann, T.J.: New Vessels for the Heart. Angiogenesis as New Treatment for Coronary Heart Disease: The Story of its Discovery and Development. Henderson, Nevada: CardioVascular BioTherapeutics Inc., 2004
• Laham, R.J., Baim, D.S.: Angiogenesis and direct myocardial revascularization. Humana Press, Totowa, NJ, 2005

Therapeutic angiogenesis

Therapeutic angiogenesis is the application of specific compounds which may inhibit or induce the creation of new blood vessels in the body in order to combat disease. The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may retard repair or some other function. Several diseases (eg. ischemic chronic wounds) are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.

The modern clinical application of the principle “angiogenesis” can be divided into two main areas: 1. Anti-angiogenic therapies (historically, research started with); 2. Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying to fight cancer and malignancies^[2][3] (because tumors, in general, are nutrition- and oxygen-dependent, thus being in need of adequate blood supply), the pro-angiogenic therapies are becoming more and more important in the search of new treatment options for cardiovascular diseases (the number one cause of death in the Western world). One of the world-wide first applications of usage of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease^[4][5][6]. Today, clinical research is ongoing in various clinical trials to promote therapeutic angiogenesis for a variety of atherosclerotic diseases, like coronary heart disease, peripheral arterial disease, wound healing disorders, etc.^[7].

Also, regarding the “mode of action”, pro-angiogenic methods can be differentiated into three main categories: 1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based therapies. There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells; 2. Risk of an undesired immune response; 3 Potential toxicity, immunogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases. In contrast, pro-angiogenic protein therapy uses well defined, precisely structured proteins, with previously defined optimal doses of the individual protein for disease states, and with well-known biological effects. On the other hand, an obstacle of protein therapy is the mode of delivery: oral, intravenous, intra-arterial, or intramuscular routes of the protein’s administration are not always as effective as desired; the therapeutic protein can be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still in an early stage of research – with many open questions regarding best cell types and dosages to use.

References

1. ^ Burri, PH (2004). "Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.". Dev Dyn. 231 (3): 474-88.
2. ^ Folkman, J, Klagsbrun, M: Angiogenetic factors. Science 235: 442-447, 1987
3. ^ Folkman J. Fighting cancer by attacking its blood supply. Sci Am. 275:150 –154, 1996
4. ^ Schumacher, B., Pecher, P., von Specht, B.U., Stegmann, T.J.: Induction of neoangiogenesis in ischemic myocardium by human growth factors. Circulation 97: 645-650, 1998
5. ^ Folkman, J.: Angiogenic therapy of the heart. Circulation 97: 628-629, 1998
6. ^ Stegmann, T.J.: A human growth factor in the induction of neoangiogenesis. Exp.Opin.Invest.Drugs 7: 2011-2015, 1998
7. ^ Wagoner, L.E., Merrill, W., Jacobs, J., Conway, G., Boehmer, J., Thomas, K., Stegmann, T.J.: Angiogenesis Protein Therapy With Human Fibroblast Growth Factor (FGF-1): Results Of A Phase I Open Label, Dose Escalation Study In Subjects With CAD Not Eligible For PCI Or CABG. Circulation 116: 443, 2007

Mechanical stimulation

Mechanical stimulation of angiogenesis is not well characterized. There is a significant amount of controversy with regard to shear stress acting on capillaries to cause angiogenesis, although current knowledge suggests that increased muscle contractions may increase angiogenesis^[8]. This may be due to an increase in the production of nitric oxide during exercise.

Stimulation by angiogenic proteins - growth factors

FGF

Further information: Fibroblast Growth Factor

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^[9]. Most are 16-18 kDa single chain peptides and display high affinity to heparin and heparan sulfate. 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 is comprised of 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, unique among all 22 members of the FGF family, 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 (hypoxic) tissues, where up regulation of FGF-receptors occurs^[10]. 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^[6].

Until now (2007), three human clinical trials have been successfully completed with FGF-1 in which the angiogenic protein was injected directly into the damaged heart muscle^{[4][11][12][7]}. Also, one additional human FGF-1 trial has been completed to promote wound healing in diabetics with chronic wounds.

Besides FGF-1, one of the most important functions of also 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, less potent than FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are important 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/cavity early in the wound healing process.

References

1. ^ Burri, PH (2004). "Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.". Dev Dyn. 231 (3): 474-88.
2. ^ Folkman, J, Klagsbrun, M: Angiogenetic factors. Science 235: 442-447, 1987
3. ^ Folkman J. Fighting cancer by attacking its blood supply. Sci Am. 275:150 –154, 1996
4. ^ ^{a b} Schumacher, B., Pecher, P., von Specht, B.U., Stegmann, T.J.: Induction of neoangiogenesis in ischemic myocardium by human growth factors. Circulation 97: 645-650, 1998
5. ^ Folkman, J.: Angiogenic therapy of the heart. Circulation 97: 628-629, 1998
6. ^ ^{a b} Stegmann, T.J.: A human growth factor in the induction of neoangiogenesis. Exp.Opin.Invest.Drugs 7: 2011-2015, 1998
7. ^ ^{a b} Wagoner, L.E., Merrill, W., Jacobs, J., Conway, G., Boehmer, J., Thomas, K., Stegmann, T.J.: Angiogenesis Protein Therapy With Human Fibroblast Growth Factor (FGF-1): Results Of A Phase I Open Label, Dose Escalation Study In Subjects With CAD Not Eligible For PCI Or CABG. Circulation 116: 443, 2007
8. ^ Prior, B. M., Yang, H. T., & Terjung, R. L. What makes vessels grow with exercise training? J App Physiol 97: 1119-28, 2004
9. ^ Ornitz, D.M., Itoh, N.: Fibroblast growth factors. Genome Biol 2: 1-12, 2001
10. ^ Blaber, M., DiSalvo, J. Thomas, K.A.: X-ray crystal structure of human acidic fibroblast growth factor. Biochemistry 35: 2086-2094, 1996
11. ^ Stegmann, T.J., Hoppert, T., Schneider, A., Popp, M., Strupp, G., Ibing, R.O., Hertel, A.: Therapeutic angiogenesis: intramyocardial growth factor delivery of FGF-1 as sole therapy in patients with chronic coronary artery disease. CVR. 2000; 1: 259-267
12. ^ Wagoner, L.E., Snavely, D.D., Conway, G.A., Hauntz, E.A., Merrill, W.H.: Intramyocardial injection of fibroblast growth factor-1 for treatment of refractory angina pectoris: the initial US experience. Circulation. 2004; 110: 395.

VEGF

VEGF (Vascular Endothelial Growth Factor) has been demonstrated to be a major contributor to angiogenesis, increasing the number of capillaries in a given network. Initial in vitro studies demonstrated that bovine capillary endothelial cells will proliferate and show signs of tube structures upon stimulation by VEGF and bFGF, although the results were more pronounced with VEGF^[13]. Upregulation of VEGF is a major component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment in vascular injuries^{[14][15][16][17]}. 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.^[8] 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, producting NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. Mechanically, VEGF is upregulated with muscle contractions as a result of increased blood flow to affected areas. The increased flow also causes a large increase in the mRNA production of VEGF receptors 1 and 2. The increase in receptor production means that muscle contractions could cause upregulation of the signaling cascade relating to angiogenesis. As part of the angiogenic signaling cascade, NO is widely considered to be a major contributor to the angiogenic response because inhibition of NO significantly reduces the effects of angiogenic growth factors. However, inhibition of NO during exercise does not inhibit angiogenesis indicating that there are other factors involved in the angiogenic response.^[8]

Angiopoietins

The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies ^[18]. Ang1 and Ang2 are protein growth factors which act by binding their receptors, Tie-1 and Tie-2; while this is somewhat controversial, it seems that cell signals are transmitted mostly by Tie-2; though some papers show physiologic signaling via Tie-1 as well. These receptors are tyrosine kinases. Thus, they can initiate cell signaling when ligand binding causes a dimerization that initiates phosphorylation on key tyrosines.

MMP

Another major contributor to angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries^[19]. These enzymes are highly regulated during the vessel formation process because destruction of the extracellular matrix would decrease the integrity of the microvasculature.^[8]

DII4

DII4 (Delta-like ligand 4), is a recently discovered protein growth factor with important angiogenic properties similar to VEGF.^[20]. Dll4 is a transmembrane ligand, for the Notch family of receptors.

Applications of Angiogenesis

Tumor angiogenesis

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A tumor consists of a population of rapidly dividing and growing cancer cells. Mutations rapidly accrue within the population. These mutations (variation) allow the cancer cells (or sub-populations of cancer cells within a tumor) to develop drug resistance and escape therapy. Tumors cannot grow beyond a certain size, generally 1-2 mm³, due to a lack of oxygen and other essential nutrients.

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. Vascular Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients -- allowing for tumor expansion. On 18 July 2007 it was discovered that cancerous cells stop producing the anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG apparently limits beta-catenin which solicits angiogenesis.^[21] Other clinicians believe that angiogenesis really serves as a waste pathway, taking away the biological end products put out by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor. Evidence now suggests that the blood vessel in a given solid tumor may in fact be mosaic vessels, comprised of endothelial cells and tumor cells. This mosaicity allows for substantial shedding of tumor cells into the vasculature. The subsequent growth of such metastases will also require a supply of nutrients and oxygen or a waste disposal pathway.

Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire 'drug resistance' to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them. Recent studies by Klagsbrun, et al. have shown, however, that endothelial cells growing within tumors do carry genetic abnormalities. Thus, tumor vessels have the theoretical potential for developing acquired resistance to drugs. This is a new area of angiogenesis research being actively pursued.

Angiogenesis research is a cutting edge field in cancer research, and recent evidence also suggests that traditional therapies, such as radiation therapy, may actually work in part by targeting the genomically stable endothelial cell compartment, rather than the genomically unstable tumor cell compartment. New blood vessel formation is a relatively fragile process, subject to disruptive interference at several levels. In short, the therapy is the selection agent which is being used to kill a cell compartment. Tumor cells evolve resistance rapidly due to rapid generation time (days) and genomic instability (variation), whereas endothelial cells are a good target because of a long generation time (months) and genomic stability (low variation).

This is an example of selection in action at the cellular level, using a selection pressure to target and differentiate between varying populations of cells. The end result is the extinction of one species or population of cells (endothelial cells), followed by the collapse of the ecosystem (the tumor) due either to nutrient deprivation or self-pollution from the destruction of necessary waste pathways.

Angiogenesis-based tumour therapy relies on natural and synthetic angiogenesis inhibitors like angiostatin, endostatin and tumstatin. These are proteins that mainly originate as specific fragments pre-existing structural proteins like collagen or plasminogen.

Recently, the 1st FDA-approved therapy targeted at angiogenesis in cancer came on the market in the US. This is a monoclonal antibody directed against an isoform of VEGF. The commercial name of this antibody is Avastin, and the therapy has been approved for use in colorectal cancer in combination with established chemotherapy.

Angiogenesis for cardiovascular disease

Angiogenesis represents an excellent therapeutic target for the treatment of cardiovascular disease. It is a potent, physiological process that underlies the natural manner in which our bodies respond to a diminution of blood supply to vital organs, namely the production of new collateral vessels to overcome the ischemic insult^[6]. Perhaps the greatest reason for these trials’ failure to achieve success is the high occurrence of the “placebo effect” in studies employing treadmill exercise test readout. Thus, even though a majority of the treated patients in these trials experience relief of such clinical symptoms such as chest pain (angina), and generally performed better on most efficacy readouts, there were enough “responders” in the blinded placebo groups to render the trial inconclusive. In addition to the placebo effect, more recent animal studies have also highlighted various factors that may inhibit an angiogenesis response including certain drugs, smoking, and hypercholesterolemia.

Although shown to be relatively safe therapies, not one angiogenic therapeutic has yet made it through the gauntlet of clinical testing required for drug approval. By capitalizing on the large database of what did and did not work in previous clinical trials, results from more recent studies with redesigned clinical protocols give renewed hope that angiogenesis therapy will be a treatment choice for sufferers of cardiovascular disease resulting from occluded and/or stenotic vessels.

Early clinical studies with protein-based therapeutics largely focused on the intravenous or intracoronary administration of a particular growth factor to stimulate angiogenesis in the affected tissue or organ. Most of these trials did not achieve statistically significant improvements in their clinical endpoints. This ultimately led to an abandonment of this approach and a widespread belief in the field that protein therapy, especially with a single agent, was not a viable option to treat ischemic cardiovascular disease. However, the failure of gene- or cell-based therapy to deliver, as of yet, a suitable treatment choice for diseases resulting from poor blood flow, has led to a resurgence of interest in returning to protein-based therapy to stimulate angiogenesis. Lessons learned from earlier protein-based studies, which indicated that intravenous or intracoronary delivery of the protein was not efficacious, have led to completed and ongoing clinical trials in which the angiogenic protein is injected directly into the beating ischemic heart.

Such localized administration of the potent angiogenic growth factor, human FGF-1, has recently given promising results in clinical trials in no-option heart patients^[22][23]. Angiogenesis was documented by angiographically visible “blushing”, and functional exercise tests were also performed on a subset of patients. The attractiveness of protein therapy is that large amounts of the therapeutic agent can be injected into the ischemic area of interest, to pharmacologically start the process of blood vessel growth and collateral arteries’ formation^[7]. In addition, from pharmacokinetic data collected from the recent FGF-1 studies in the human heart, it appears that FGF-1, once it exits the heart is cleared in less than three hours from the circulation. This would presumably prevent FGF-1 from stimulating unwanted angiogenesis in other tissues of the bodies where it could potentially cause harm, such as the retina and in the kidneys. No serious adverse events have yet to be noted in any of the completed or ongoing clinical trials in which the FGF-1 protein is utilized as the therapeutic agent tom stimulate angiogenesis^[7][24].

Left: Angiographic "blushing" following FGF-1 injection into the human heart. Right, measurements of pixel density in angiograms ("gray-value-analysis") indicating a threefold increase in vessel density in the treated human myocardium (3 months & 3 years).

Improvement in myocardial perfusion (blood supply) after FGF-1 treatment as demonstrated by SPECT imaging (single photon emission computed tomography).

Exercise

Angiogenesis is generally associated with aerobic exercise and endurance exercise. While arteriogenesis produces network changes that allow for a large increase in the amount of total flow in a network, angiogenesis causes changes that allow for greater nutrient delivery over a long period of time. Capillaries are designed to provide maximum nutrient delivery efficiency so an increase in the number of capillaries allows the network to deliver more nutrients in the same amount of time. A greater number of capillaries also allows for greater oxygen exchange in the network. This is vitally important to endurance training because it allows a person to continue training for an extended period of time. However, no experimental evidence exists to suggest that increased capillarity is required in endurance exercise to increase the maximum oxygen delivery.^[8]

Macular degeneration

Overexpression of VEGF causes increased permeability in blood vessels in addition to stimulating angiogenesis. In wet macular degeneration VEGF causes proliferation of capillaries into the retina. Since the increase in angiogenesis also causes edema, blood and other retinal fluids leak into the retina causing loss of vision. A novel treatment of this disease is to use a VEGF inhibiting siRNA to stop the main signaling cascade for angiogenesis.

See also

• antiangiogenic therapy
• arteriogenesis
• endurance exercise
• aerobic exercise

References

1. ^ Burri, PH (2004). "Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.". Dev Dyn. 231 (3): 474-88.
2. ^ Folkman, J, Klagsbrun, M: Angiogenetic factors. Science 235: 442-447, 1987
3. ^ Folkman J. Fighting cancer by attacking its blood supply. Sci Am. 275:150 –154, 1996
4. ^ ^{a b} Schumacher, B., Pecher, P., von Specht, B.U., Stegmann, T.J.: Induction of neoangiogenesis in ischemic myocardium by human growth factors. Circulation 97: 645-650, 1998
5. ^ Folkman, J.: Angiogenic therapy of the heart. Circulation 97: 628-629, 1998
6. ^ ^{a b c} Stegmann, T.J.: A human growth factor in the induction of neoangiogenesis. Exp.Opin.Invest.Drugs 7: 2011-2015, 1998
7. ^ ^{a b c d} Wagoner, L.E., Merrill, W., Jacobs, J., Conway, G., Boehmer, J., Thomas, K., Stegmann, T.J.: Angiogenesis Protein Therapy With Human Fibroblast Growth Factor (FGF-1): Results Of A Phase I Open Label, Dose Escalation Study In Subjects With CAD Not Eligible For PCI Or CABG. Circulation 116: 443, 2007
8. ^ ^{a b c d e} Prior, B. M., Yang, H. T., & Terjung, R. L. What makes vessels grow with exercise training? J App Physiol 97: 1119-28, 2004
9. ^ Ornitz, D.M., Itoh, N.: Fibroblast growth factors. Genome Biol 2: 1-12, 2001
10. ^ Blaber, M., DiSalvo, J. Thomas, K.A.: X-ray crystal structure of human acidic fibroblast growth factor. Biochemistry 35: 2086-2094, 1996
11. ^ Stegmann, T.J., Hoppert, T., Schneider, A., Popp, M., Strupp, G., Ibing, R.O., Hertel, A.: Therapeutic angiogenesis: intramyocardial growth factor delivery of FGF-1 as sole therapy in patients with chronic coronary artery disease. CVR. 2000; 1: 259-267
12. ^ Wagoner, L.E., Snavely, D.D., Conway, G.A., Hauntz, E.A., Merrill, W.H.: Intramyocardial injection of fibroblast growth factor-1 for treatment of refractory angina pectoris: the initial US experience. Circulation. 2004; 110: 395.
13. ^ Goto, F., Goto, K., Weindel, K., & Folkman, J. Synergistic effects of vascular endothelial growth-factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Inves 69: 508-17, 1993
14. ^ Ding, Y. H., Luan, X. D., Li, J., Rafols, J. A., Guthinkonda, M., & Diaz, F. G. et al. Exercise-induced overexpression of angiogenic factors and reduction of ischemia/reperfusion injury in stroke. Curr Neurovasc Res 1: 411-20, 2004
15. ^ Gavin, T. P., Robinson, C. B., Yeager, R. C., England, J. A., Nifong, L. W., & Hickner, R. C. Angiogenic growth factor response to acute systemic exercise in human skeletal muscle. J App Physiol 96: 19-24, 2004
16. ^ Kraus, R. M., Stallings, H. W., Yeager, R. C., & Gavin, T. P. Circulating plasma VEGF response to exercise in sedentary and endurance-trained men. J App Physiol 96: 1445-50, 2004.
17. ^ Lloyd, P. G., Prior, B. M., Yang, H. T., & Terjung, R. L. Angiogenic growth factor expression in rat skeletal muscle in response to exercise training. Am J Physiol Heart Circ Physiol 284: 1668-78, 2003.
18. ^ Thurston G. Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis. Cell Tissue Res. 2003 Oct;314(1):61-8. Epub 2003 Aug 12.
19. ^ Haas, T. L., Milkiewicz, M., Davis, S. J., Zhou, A. L., Egginton, S., Brown, M. D., Madri, J. A., Hudlicka, O. Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle. Am J Physiol Heart Circ Physiol 279: H1540-H1547, 2000
20. ^ , Lobov IB, Renard RA, Papadopoulos N, Gale NW, Thurston G, Yancopoulos GD, Wiegand SJ. (2007) Proc Natl Acad Sci U S A. 2007 104(9):3219-24. Epub 2007 Feb 12.
21. ^ [1]
22. ^ Stegmann, T.J., Hoppert, T., Schneider, A., Popp, M., Strupp, G., Ibing, R.O., Hertel, A.: Therapeutic angiogenesis: intramyocardial growth factor delivery of FGF-1 as sole therapy in patients with chronic coronary artery disease. CVR.1: 259-267, 2000
23. ^ Wagoner, L.E, Snavely, D.D., Conway, G.A., Hauntz, E.A., Merrill, W.H.: Intramyocardial injection of fibroblast growth factor-1 for treatment of refractory angina pectoris: the initial US experience. Circulation; 110: 395, 2004
24. ^ Stegmann TJ. New Vessels for the Heart. Angiogenesis as New Treatment for Coronary Heart Disease: The Story of its Discovery and Development. Henderson, Nevada: CardioVascular BioTherapeutics Inc., 2004