(Circulation. 1996;94:1496-1498.)
© 1996 American Heart Association, Inc.
Articles |
Use of Vascular Endothelial Growth Factor for Therapeutic Angiogenesis
the Vascular Cell Biology Laboratory, Texas Heart Institute, Houston, and the Department of Internal Medicine, Division of Cardiology, University of Texas Medical School, Houston.
Correspondence to David Engler, PhD, Texas Heart Institute, Vascular Cell Biology, PO Box 20345 MC 2-255, Houston, TX 77225-0345. E-mail dengler@biost1.thi.tmc.edu.
Key Words: Editorials • growth substances • receptors • revascularization
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Introduction |
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 Top Introduction References |
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The term "therapeutic angiogenesis" was coined to describe intervention used to treat or prevent pathological clinical situations characterized by local hypovascularity by stimulating or inducing neovascularization.1 Angiogenesis is defined as the formation of new blood vessels from preexisting vessels by the process of cellular outgrowth. Angiogenesis differs from vasculogenesis, which is an embryonic phenomenon in which new blood vessels are formed de novo from blood islands composed of committed stem cells. Therefore, angiogenesis accounts for all new vessel growth after embryonic life and is a normal function of such processes as injury repair and the female reproductive cycle. The angiogenic process requires a concerted series of events. These include the migration and proliferation of endothelial cells in and away from the original microvasculature, the breakdown of basement membranes and the controlled expression of proteolytic enzymes that can degrade extracellular matrices, the reassembly of new extracellular matrix, and endothelial tube formation. Under certain circumstances, insufficient angiogenesis can lead to such problems as ulcers that do not heal and myocardial infarction. Conversely, many pathologies are complicated by unregulated angiogenesis and are typified by cancer, diabetic retinopathy, and atherosclerosis itself (see below).
The normal process of angiogenesis is controlled by the balance of proangiogenic and antiangiogenic molecules that are spatially and temporally regulated in vivo (reviewed in Reference 2 and references cited there). In addition to VEGF, proangiogenic molecules include members of the FGF family, transforming growth factor-ß, tumor necrosis factor-
, platelet-derived growth factor-BB, and others. These soluble molecules exert their angiogenic stimuli by coupling to cell surface receptor molecules that span the plasma membrane and trigger the requisite functions within endothelial cells through signaling cascades. Recently, much interest has been placed on the role of VEGF (also known as vascular permeability factor) in angiogenesis because the receptors that control the intracellular signaling elicited by this molecule have been reported to be specific to endothelial cells, whereas the receptors for most other proangiogenic molecules are more ubiquitous in their cellular distribution.
Four forms of VEGF arise from alternative splicing of the mRNA from a single gene. These forms code for protein molecules of 121, 165, 189, and 206 amino acids. All forms are secreted glycoproteins that homodimerize and, except for VEGF121, are capable of binding heparin or cell surface heparan sulfate proteoglycans. The two larger forms apparently stay membrane bound, presumably because of their tighter binding to heparan sulfate proteoglycans. VEGF165 is the predominant form, secreted by a variety of normal and transformed cells; VEGF206 is the rarest.3 All forms are mitogenic toward vascular endothelial cells and induce vascular permeabilization of blood vessels.
VEGF isoforms are capable of binding to two characterized VEGFRs on the surface of endothelial cells that function as protein-tyrosine kinases. These are designated as Flt-1/VEGFR-1 (fms–like–tyrosine kinase4 ) and KDR/VEGFR-2 (for kinase insert domain–containing receptor5 ). VEGF165 is known to bind to both VEGF receptor forms, whereas VEGF121 has only recently been shown to be specific for the KDR/VEGFR-2.6 Although these receptors are found primarily on endothelial cells in vivo, some studies have identified these receptors on certain nonendothelial cells in culture. Most notable are human melanoma cells7 that respond in a mitogenic fashion to exogenous VEGF addition. A third receptor tyrosine kinase named Flt-4 is related to the VEGF receptors but does not bind VEGF. Its distribution is restricted to the lymphatic endothelia. Recently, an apparently novel VEGFR has been identified on breast tumor cells8 that specifically binds VEGF165. It is not yet clear whether the display of these receptors is an artifact of culture conditions or whether the VEGFR family may be more widely displayed than originally thought under both normal and pathological conditions.
Other VEGF-like molecules have been identified. Placenta growth factor is expressed in the placenta and in vascular endothelial cells and is capable of binding the Flt-1/VEGFR-1 receptor. Recently, a molecule identified as VEGF-B9 was cloned and shown to form cell surface–bound homodimers. It is also capable of forming heterodimers with VEGF165, keeping it localized to the cell surface. It is not yet known whether VEGF-B is capable of binding to either Flt-1/VEGFR-1 or KDR/VEGFR-2 or whether it has a specific receptor. VEGF-C (also known as VEGF-related protein10 11 ) also has been cloned recently. VEGF-C has been identified as the specific ligand for Flt-4 in the lymphatic endothelia. VEGF-C also is capable of binding KDR/VEGFR-2 and may play a role in the control of vascular endothelia.
It is thought that signaling downstream of the KDR/VEGFR-2 receptor is important in controlling most of the effects of VEGF on angiogenesis. Waltenberger et al12 have shown that cells lacking endogenous VEGFRs, when transfected with KDR/VEGFR-2 expression constructs and stimulated with VEGF, show changes in cell morphology, actin reorganization, membrane ruffling, chemotaxis, and mitogenicity, whereas Flt-1/VEGFR-1–expressing cells lacked such responses on VEGF addition. Homologous recombination studies in mice have clearly shown that both receptors are important in vasculogenesis although they differ in temporal importance to the process. Mice made deficient in Flt-1/VEGFR-1 activity by mutation induced by homologous recombination still produced endothelial cells during vasculogenesis but assembled them into abnormal vascular channels during development and produced lethality.13 In contrast, similar mice made deficient in the mouse homologue to KDR/VEGFR-2 (Flk-1) died earlier in development owing to defects in the development of hematopoietic progenitors and blood islands that form the endothelial cells.14
The importance of VEGF in angiogenesis is further underscored by its regulation at the transcriptional and posttranscriptional levels under conditions of hypoxia. Hypoxia is a major driving force for angiogenesis. Hypoxic conditions induce increased transcription of the mRNA for VEGF and increase its stability in vascular smooth muscle cells. It is also known that vascular endothelial cells are capable of enhanced production of VEGF under hypoxic conditions.15 In this issue, Waltenberger et al16 have provided further evidence in support of using VEGF in a therapeutic angiogenesis scenario by showing that the receptor most likely to increase angiogenesis in ischemic tissue, KDR/VEGFR-2, is also functionally upregulated in response to hypoxia. They describe differences in the mode of this upregulation from that observed for VEGF, namely that the mRNA levels are not increased in response to hypoxia (in fact, they are decreased), but protein levels of the functional receptor are augmented in an as-yet-uncharacterized posttranscriptional regulatory manner. Previous work by others17 also has indicated that a factor induced by hypoxia and produced by both vascular smooth muscle cells and vascular endothelial cells is capable of causing an upregulation of KDR/VEGFR-2 protein on the surface of endothelial cells. The mRNA for both VEGF and KDR/VEGFR-2 also has been reported to be upregulated in the lung tissue of rats chronically exposed to hypoxic conditions.18
The functional upregulation of the KDR/VEGFR-2 under these conditions has further ramifications for antiangiogenesis, ie, therapeutic modalities concerned with inhibiting angiogenesis. It is well known that solid tumor growth is regulated by angiogenesis and is driven in part by local hypoxia within the tumor. One recent study demonstrated, at least for colon cancer, that the expression of both VEGF and KDR/VEGFR-2 correlated not only with tumor proliferation rate and extent of vascularization but also with metastasis. The same was not true for another angiogenic factor (bFGF) and its receptors.19 Such observations provide evidence for using the KDR/VEGFR-2 as a potential target for antiangiogenesis cancer therapy.
The further possibility that upregulation of KDR/VEGFR-2 may function in concert with VEGF as an adaptation to hypoxic stress for enhancing endothelial cell survival under ischemic conditions is not without precedent. Recent data suggest that VEGF treatment of normal hematopoietic cells can enhance survival of these cells by the inhibition of apoptosis caused by ionizing radiation.20 This effect is presumably mediated through KDR/VEGFR-2, which is displayed on these cells. Thus, VEGF therapy has the potential to go beyond the stimulation of angiogenesis to draw on its ability to act as a survival-enhancing agent and be an adjuvant to current therapies already in use for various pathologies.
Recent clinical studies carried out in patients with peripheral artery disease21 underscore the potential usefulness of VEGF angiogenic therapy. However, although it is certain that VEGF is an important mediator of angiogenesis both in vitro and in vivo, recent studies have suggested that it alone may be insufficient for complete angiogenesis, at least in some cases. VEGF (or bFGF) alone was insufficient to stimulate the in vitro formation of capillarylike structures formed by human microvascular endothelial cells in fibrin matrices without the direct addition or indirect stimulation by tumor necrosis factor- of urokinase-type plasminogen activator.22 This recent finding, however, may be specific to species or vascular bed, because other studies have shown VEGF to be capable of directly inducing urokinase-type plasminogen activator production in bovine microvascular endothelial cells.23 Other studies also have shown a potent synergistic effect of VEGF and bFGF on angiogenesis in culture23 and in animal models.24
Although enthusiasm for therapeutic angiogenesis using VEGF is understandably great at the present time, it must be tempered with a thorough understanding of all the potential risks involved. With respect to VEGF- or KDR/VEGFR-2–related therapies, one must be mindful of the ever-increasing number of cell types being identified as displaying what was once thought to be an "endothelial cell–specific" receptor. This will affect the type and the delivery strategy of the contemplated therapy. Additional thought must be given to "working doses" of the angiogenic factor, with the desired end point and preexisting conditions in a given environment for the therapy of interest taken into account. The case can be exemplified by one potential problem in inducing angiogenesis to treat coronary occlusions. In certain atherosclerotic plaques, the tiny vessels within the plaque comprising the vasa vasorum, a product of angiogenesis in its own right, might be rendered more permeable by VEGF stimulation and hemorrhage on the very VEGF addition originally given to circumvent the blockage. This, in turn, could cause thrombosis and lead directly to myocardial infarction. This scenario is supported by one recent study25 that demonstrated that in some restenotic lesions, the total endothelial cell replication index in the vasa vasorum was as high as 43.5% without exogenous VEGF addition. These vascularized plaques consistently included macrophages, which quite possibly could make them additionally prone to rupture on VEGF treatment and initiate clot formation. The use of VEGF for therapeutic angiogenesis holds promise for clinical situations in which it can be safely assumed that the formation of new vessels involved in the overall reparative process (including tissue remodeling or preservation of important ischemic tissue) will outweigh the potentially harmful angiogenesis that can promote disease or lead to further tissue damage.
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Selected Abbreviations and Acronyms |
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editor or the American Heart Association.
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References |
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TopIntroductionReferences |
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