by: Navonil Ghosh
Abstract
Angiogenesis, the growth of new blood vessels from preexisting vasculature, is a significant physiological process in living organisms. Vascular endothelial growth factors, or VEGF, and associated receptors are key effectors during the formation of blood vessels. Because any defects or abnormalities in vessel formation can result in a variety of vascular diseases and disorders, an intimate understanding of the developmental pathways that regulate angiogenesis could elucidate the causes of these diseases and reveal possible treatments or preventative measures. Thus, scientists are exploring a number of angiogenic signaling pathways and effectors of these pathways, particularly VEGF. By manipulating these pathways and analyzing the resulting phenotypic effects on blood vessel formation, researchers can determine the effectiveness of angiogenic regulatory molecules as therapeutic agents for vascular disorders.
Introduction
The vasculature of vertebrate organisms is regulated by a number of signaling pathways that affect vascular endothelial cells, which comprise the inner lining of blood vessels. These pathways govern both the growth and patterning of blood vessel formation. Any disruption to the developmental cues necessary for proper growth can result in defective vascular formation that can lead to a number of diseases including psoriasis, rheumatoid arthritis, and cancer. Because the blood vessels of the vascular system are intricately linked to the survival of all vertebrates, a vast number of clinical and developmental studies have focused on angiogenesis.
Although the term was first used by Dr. John Hunter in 1787 to describe blood vessel growth in reindeer antlers, angiogenesis entails the formation of new capillary blood vessels in all vertebrates and is a major area of developmental study.1 In healthy vertebrate organisms, angiogenesis is a key component to healing wounds and restoring circulation to damaged tissue. The process is controlled in a healthy organism by a series of angiogenesis-stimulating growth factors and angiogenesis inhibitors, which serve as the “on” and “off” switches for blood vessel growth.2 The angiogenic process was thus proposed to have a therapeutic role by means of new vessel induction in damaged tissue or inhibition of vascular formation in pathological pathways involving abnormal vessel growth. According to the Angiogenesis Foundation, approximately 500 million patients in Western nations alone could benefit from either proangiogenic or antiangiogenic therapy. As a result, over $4 billion in medical research has been devoted to developing angiogenic-based therapeutic drugs.1
Angiogenic Vessel Formation
Angiogenesis is dependent on a signaling cascade to induce vascular development (Fig. 1).1 The first step of the angiogenic process is the release of angiogenic growth factors that bind to the membrane-bound receptors of nearby vascular endothelial cells. These activated endothelial cells then start producing and secreting enzymes that degrade the basement membrane, a sheath-like covering of blood vessels. They also initiate an intracellular signaling cascade that prompts the formation of other building blocks of endothelial cell formation. The endothelial cells divide and pass through the dissolved openings of the basement membrane to where the new vessel will form. The sprouting of the newly developing vessel is aided by adhesion molecules known as integrins that bind and help pull the vessel into place. Meanwhile, other enzymes such as matrix metalloproteinases degrade the surrounding extracellular matrix to make room for the growing vessel. The endothelial cells then join together to form individual capillary sprouts, which are stabilized by smooth muscle cells and pericyte muscle cells. Blood circulation begins when the individual tubes join together to form blood vessel loops.
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is one of the most critical effectors involved in angiogenesis. VEGF, a family of tyrosine kinases, stimulates a variety of signaling pathways that regulate the growth of endothelial cells.3 The VEGF system involves a series of ligands and receptors. The secreted ligands include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF and the receptors include VEGF-R1, VEGF-R2, VEGF-R3. Through autophosphorylation by the tyrosine kinases of the VEGF family, the VEGF receptors activate cellular signaling pathways that ultimately control migration, survival, and proliferation of vascular endothelial cells. Because VEGF plays such an intimate role in angiogenesis, it is highly utilized in a variety of scientific studies involving other angiogenic factors.4 A recent study, for example, shows that angiomodulin, a modulator of VEGF-dependent vascular formation, has increased expression in human tumor tissue.5 Although further examination of angiomodulin’s role in cancer progression is needed, this could mark a step in evaluating angiogenic pathways in a therapeutic role for cancer.
The Zebrafish Model
There are a variety of in vivo models available for angiogenesis (Table 1).6 The zebrafish model organism, Danio rerio, is particularly well-suited for the analysis of developing vasculature structures for a variety of reasons. It produces a large clutch size of transparent, external embryos that allow easy, accessible observation from an early age.7 Furthermore, the rapid development of zebrafish embryos also allows for timely analysis and easy reproduction of experimental results. The genetic pathways of zebrafish share many angiogenic mechanisms and genes with other vertebrates, and because these pathways can be easily manipulated, the zebrafish can serve as a useful tool for analyzing the development of vasculature in vertebrate organisms in general.8 Moreover, the vasculature itself can be easily observed through the use of transgenics, allowing the intersegmental, subintestinal, and cranial vessels of the organism to be easily examined (Fig 2.).9 By virtue of these advantages, the zebrafish is a highly used model organism by scientists studying the processes that regulate blood vessel formation.
In order to reproduce defects in the vasculature of the zebrafish model organism, scientists rely on a number of effective assays and experimental procedures. In order to visibly examine the blood vasculature of zebrafish, transgenic zebrafish with fluorescent vessels have been developed. Fluorescent expression through the insertion of the green fluorescent protein (GFP) construct into fli-eGFP, mTie2-GFP, and flk-GFP promoters, which are expressed in vascular endothelial cells, allows the entire vasculature of the organism to be visible.8 Microinjection of mRNA and morpholinos can be used to examine a target gene’s role in vascular development by showing the effects of altering normal gene expression.7 Injecting mRNA overexpresses the target gene, while the morpholinos block translation to knockdown the gene. Injections into transgenic zebrafish allow the effects of overexpression and knockdown to be examined in great detail. Further genetic analysis can be done through in situ hybridizations, which uses antibodies to stain a complementary strand of RNA that binds to a target gene. This allows for the identification of changes in the expression of other genes through alteration of the original target gene.
Angiogenic-Dependent Diseases
Angiogenesis-based pathological conditions can occur when the body loses control over the angiogenic process and blood vessel formation progresses either excessively or insufficiently .1 In diseases dependent on excessive angiogenesis, diseased cells release excessive amounts of angiogenic growth factors that cause abnormal proliferation of the endothelial cells that make up newly developing vessels. These excessively sprouting blood vessels damage healthy tissue while continuing to nourish the diseased tissue. Conditions related to an overgrowth of blood vessels include macular degeneration, diabetic blindness, and psoriasis. Abnormal vessels can even allow cancerous growths to metastasize by providing the means for tumor cells to reach healthy organs. In the case of insufficient angiogenic vessel growth, the body cannot produce enough angiogenic growth factors for proper vessel formation. The lack of adequate blood vessel growth affects tissue perfusion and can cause necrosis. Insufficient angiogenesis is involved with both coronary artery disease and stroke.
Angiogenic Therapy and Inhibition
Because so many pathological conditions have a tie to abnormal blood vessel formation, scientists have proposed that angiogenesis may have therapeutic purposes. Therapeutic angiogenesis generally involves turning the genes controlling the process, or using angiogenesis – stimulating growth factors to allow for the growth of new blood vessels.1 Angiogenic stimulation targets vascular endothelial cells by using cytokine growth factors, such as those of the VEGF family. The resulting increase in endothelial cell proliferation enhances vessel formation and blood profusion to damaged, or ischemic, tissue. This proangiogenic stimulation is induced in order to enhance blood flow to injured tissue, transport survival factors to areas in need of tissue repair, activate populations of regenerative stem cells, and ultimately restore tissue to its normal state.
A number of clinical drugs and interventions have been developed to treat pathologies through therapeutic angiogenesis. The three diseases most commonly treated using angiogenic therapy are chronic wounds, such as arterial or venous ulcers, peripheral arterial disease, and ischemic heart disease.1 To treat these diseases, angiogenesis can be stimulated through therapeutic angiogenic drugs. An FDA-approved growth-factor based drug known as rhPDGF is a recombinant protein medication used to treat diabetic ulcers. Systems such as autologel or SmartPReP allow for the treatment of injured tissue using autologous blood samples, which contain the natural growth factors and cytokines needed to stimulate healing through angiogensis. 10, 11 Early clinical trials of other angiogenic stimulators have shown evidence of increased blood flow and function of the left ventricle. This is a promising therapeutic tool for treating coronary heart disease. Angiogenesis can also be promoted without relying on drugs or medication. Negative pressure wound therapy stimulates blood vessel growth through micro-deformations of tissue that respond with signal transduction leading to epithelial proliferation. Other methods include the MIST ultrasound, a low-frequency and low-intensity procedure that stimulates endothelial cells and enhances blood perfusion, and Hyperbaric Oxygen (HBO), which increases expression of VEGF and endothelial progenitor cells.12, 1
Inhibition of angiogenesis can also have therapeutic properties, for it can prevent feeding of diseased tissue such as cancerous tumors. Anti-angiogenic agents inhibit cell signaling pathways necessary for vessel growth. These inhibitors include three major varieties: monoclonal antibodies, tyrosine kinase inhibitors, and inhibitors of mammalian targets of rapamycin (mTOR).1 Monoclonal antibodies bind to VEGF to prevent it from binding in turn to VEGF receptors, thus interrupting the proliferation of endothelial cells. Small molecule tyrosine kinase inhibitors interrupt pathways involving a number of tyrosine kinase growth factor receptors, including VEGF receptors. mTOR inhibitors intercept the PI3/AKT pathway necessary for tumor proliferation. 13 All three methods act by inhibiting a pathway necessary for endothelial proliferation and thus prevent angiogenic nourishment of diseased or cancerous tissue.
Conclusions
Therapeutic proangiogenic and antiangiogenic applications have shown potential in alleviating a number of vascular-based pathologies. The zebrafish developmental model remains a valuable tool for analyzing vascular formation and potential angiogenic therapies. As our understanding of angiogenesis increases, advancements in therapeutic uses for the process will have an important impact on clinical treatment of vascular pathologies.
Acknowledgements
I would like to thank Dr. Daniel Wagner for introducing me to the angiogenic and zebrafish research. I would also like to thank Jane Li, Casey O’Grady, Eric Kim, and Dr. Amina Qutub for their feedback and advice upon editing my article.
References
1. “Understanding Angiogenesis”. The Angiogenesis Foundation. 10 July 2009. .
2. “Angiogenesis Overview”. Angioplasty.Org. Venture Digital LLC, 1996-2009. .
3. Rahimi, Nader. Vascular endothelial growth factor receptors: Molecular mechanisms of activation and therapeutic potentials. Exp Eye Res. 2006 November; 83(5): 1005–1016. Published online 2006 May 19.
4. Sato, Thomas. Emerging concept in angiogenesis: specification of arterial and venous endothelial cells. Br J Pharmacol. 2003 October; 140(4): 611–613. Published online 2003 October 15.
5. Hooper, A.T., Shmelkov, S.V., Gupta, et al. Angiomodulin Is a Specific Marker of Vasculature and Regulates Vascular Endothelial Growth Factor-A-Dependent Neoangiogenesis. Circ. Res. 105(2):201-208 (Journal).
6. K. Norrby. In vivo models of angiogenesis. J. Cell. Mol. Med. Vol 10, No 3, 2006 pp. 588-612.
7. Kameha R Kidd and Brant M Weinstein. Fishing for novel angiogenic therapies. Br J Pharmacol. 2003 October; 140(4): 585- 594. Published online 2003 October 15.
8. Carolyn A. Staton, Malcolm W. R. Reed and Nicola J. Brown. A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Pathology. Volume 90, Issue 3, Pages 195-221. Published Online: 11 May 2009.
9. Isogai, S., Horiguchi, M., and Weinstein, B. M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Developmental Biology, Volume 230, pp. 278-301.
10. “The Autologel System – Natural Healing Wound Care using platelet rich plasma (PRP).” Cytomedix, 2009. .
11. “Harvest Technologies – Overview.” Harvest Technologies, Corp., 2009. .
12. Latham, Emi. “Hyperbaric Oxygen Therapy.” eMedicine, 1994-2010. .
13. Sandrine Faivre, Guido Kroemer & Eric Raymond. Current development of mTOR inhibitors as anticancer agents. Nature Reviews Drug Discovery 5, 671-688. August 2006.
14. Garzon, M.C., J.T. Huang, O. Enjolras, and I.J. Frieden, Vascular malformations: Part I. J Am Acad Dermatol, 2007. 56(3): p. 353- 70.
15. Thurston, G. and Kitajewski, J. VEGF and Delta-Notch: interacting signalling pathways in tumour angiogenesis. Br J Cancer. 2008 October 21; 99(8): 1204–1209. Published online 2008 September 30.
16. Brant M. Weinstein. What guides early embryonic vessel formation? Developmental Dynamics. Volume 215, Issue 1, Pages 2-11. Published Online: 21 Sep 1999.
17. Young Ryun Cha, Brant M. Weinstein. Visualization and experimental analysis of blood vessel formation using transgenic zebrafish. Volume 81, Issue 4, Pages 286-296. Published Online: 28 Jan 2008.
Navonil Ghosh is a sophomore from Hanszen College majoring in Biochemistry and Cell Biology. Under the supervision of Dr. Daniel Wagner of the Biochemistry and Cell Biology Department at Rice University, Ghosh is investigating the role of the aggf1 gene in angiogenesis. Angiogenic Factor with G patch and FHA domains 1 (AGGF1) has been associated with the VEGF pathway, as well as a vascular birth defect known as Klippel-Trenaunay-Weber Syndrome. Thus, aggf1 is hypothesized as an important regulator of vascularization in zebrafish. Ghosh is analyzing the effects of overexpression and knockdown of aggf1 on the vasculature of the zebrafish model in order to better understand the role of aggf1 in angiogenesis and evaluate its therapeutic potential.