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.
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.
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.
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 (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 worldwide 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. 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..
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.
|FGF||Promotes proliferation & differentiation of endothelial cells, smooth muscle cells, and fibroblasts|
|VEGFR and NRP-1||Integrate survival signals|
|Ang1 and Tie2||Stabilize vessels|
|PDGF (BB-homodimer) and PDGFR||recruit smooth muscle cells|
|TGF-β, endoglin and TGF-β receptors||↑extracellular matrix production|
|Integrins αVβ3, αVβ5 (?) and α5β1||Bind matrix macromolecules and proteinases|
|VE-cadherin and CD31||endothelial junctional molecules|
|ephrin||Determine formation of arteries or veins|
|plasminogen activators||remodels extracellular matrix, releases and activates growth factors|
|plasminogen activator inhibitor-1||stabilizes nearby vessels|
|NOS and COX-2|
|AC133||regulates angioblast differentiation|
|Id1/Id3||Regulates endothalial transdifferentiation|
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. 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 composed 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. 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.
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. 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.
The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies . 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.
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. 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, composed 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.
If one reviews in detail the various published angiogenesis clinical trials, it can be realized that most of these trials had success in achieving various secondary or supportive endpoints, but failed when attempting to demonstrate a statistically significant improvement in exercise performance, typically done by a treadmill exercise test. 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.
These failures suggested that either these are the wrong molecular targets to induce neovascularization, that they can only be effectively utilized if formulated and administered correctly, or that their presentation in the context of the overall cellular microenvironment may play a vital role in their utility. It may be necessary to present these proteins in a way that mimics natural signaling events, including the concentration, spatial and temporal profiles, and their simultaneous or sequential presentation with other appropriate factors.
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. 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. 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.
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).