(Circulation. 1996;94:3062-3064.)
© 1996 American Heart Association, Inc.
Articles |
A Little VEGF Goes a Long Way
Therapeutic Angiogenesis by Direct Injection of Vascular Endothelial Growth Factor–Encoding Plasmid DNA
the Departments of Pathology and Cell Biology, Baylor College of Medicine, Houston, Tex.
Correspondence to Dr Mark Majesky, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail mmajesky@bcm.tmc.edu.
Key Words: Editorials • angiogenesis • ischemia • genes • peripheral vascular disease
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Introduction |
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 Top Introduction VEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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Local production of small amounts of a potent, secreted growth factor with built-in specificity for endothelial cells may bypass the need for highly efficient gene transfer vectors and elaborate treatment protocols to produce effective therapeutic angiogenesis in ischemic peripheral vascular disease. That is the message of a report by Tsurumi et al that appears in this issue of Circulation1 in which naked plasmid DNA encoding the 165–amino acid secreted form of human vascular endothelial growth factor (hVEGF-165) is introduced by direct intramuscular injection into ischemic rabbit hindlimbs. Thirty days after injection of 100 µg hVEGF-165 plasmid DNA at each of five sites within ischemic hindlimb muscles, increased collateral vessel formation, improved blood flow, and an increased number of capillary profiles could be demonstrated compared with control hindlimbs that received injections of plasmid DNA only. The simplicity of the gene transfer approach described in this study suggests a potentially useful alternative for patients with extensive peripheral vascular disease in whom intravascular angioplasty catheter–based gene delivery methods are inadvisable or not possible.
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VEGF: An Endothelial Cell–Specific Ligand and Receptor Signaling Pair |
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TopIntroductionVEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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VEGF was discovered in the early 1980s by Dvorak and coworkers as a factor that made blood vessels leaky; hence, it was given the name of vascular permeability factor (VPF).2 3 Then, in the late 1980s, several groups showed that VEGF/VPF stimulated endothelial cell migration and replication and was a potent angiogenic factor in vivo.4 5 6 VEGF is a heat-stable, 46-kD dimeric protein with structural similarity to placenta growth factor (PlGF) (
53% amino acid identity) and more distant homology (18% to 20%, including all eight cysteines) to the platelet-derived growth factor family (for review, see Reference 6). VEGF is made by many different cell types, and its production is upregulated in hypoxic tissues.7 Indeed, a 28-bp element has been identified in the promoter region of the rat VEGF gene that mediates hypoxia-inducible transcription in transient transfection assays.8 The hypoxia-response element in the VEGF promoter has sequence and protein-binding properties very similar to the hypoxia-inducible factor-1 binding site within the erythropoietin enhancer.8 The biological activities of VEGF are mediated by two transmembrane receptor tyrosine kinases termed flt-1 and flk-1/KDR that are expressed predominantly on vascular endothelial cells and their embryonic progenitors (angioblasts). The two receptors differ in their intrinsic affinity for VEGF. Flt-1 is a higher-affinity receptor (Kd=16 to 30 pmol/L), whereas flk-1/KDR has a somewhat lower affinity for VEGF (3 to 500 pmol/L). The expression of VEGF receptors is also upregulated by hypoxia.7 |
VEGF in Embryonic Vasculogenesis |
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TopIntroductionVEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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A critical role for the VEGF receptors flk-1/KDR and flt-1 in vasculogenesis during embryonic development was recently established by targeted gene-deletion studies in mice.9 10 Flk-1–deficient embryos never developed a vascular system and were nearly devoid of hemangioblast derivatives, ie, endothelial cells and blood cells.9 Although it was generally assumed that the dramatic phenotype of VEGF receptor–deficient mice was evidence for an essential role of VEGF in angioblast differentiation and vascular morphogenesis, the ability of flt-1 receptors to also bind the structurally related cytokine PlGF meant that the precise role of VEGF in vasculogenesis remained uncertain. Earlier this year, two independent reports11 12 both demonstrated an absolute requirement for VEGF in development of the vascular system in the mouse embryo. Remarkably, both studies found that VEGF-deficient embryos harboring only one inactivated VEGF allele exhibited a heterozygous lethal phenotype, an unprecedented finding for an autosomal gene inactivation, indicative of an extremely tight concentration dependence for vessel formation by VEGF. Because in the case of VEGF receptors, embryonic lethality was observed in homozygous-deficient but not in heterozygous-deficient embryos, it would seem that VEGF receptors are normally present in excess and that nascent endothelial cells are highly sensitive to variations in local concentrations of VEGF for new vessel formation in the embryo. This prediction appears to be borne out by the studies of Drake and Little,13 who microinjected VEGF-165 into the normally avascular space between the endoderm and the splanchnic mesoderm in 5-somite-stage quail embryos. These injections of VEGF resulted in (1) formation of extra, malformed vessels that joined the dorsal aortae and outflow tract directly to the inflow regions of the developing heart and (2) excessive fusion of vessels to produce abnormally large lumens and altered branching patterns. Similar findings were reported by Flamme et al14 using a retroviral vector to express VEGF-165 in various places and times in quail embryogenesis. Interestingly, Flamme et al14 found that expression of the endogenous flk-1 gene was upregulated only in those areas where VEGF was overexpressed. This suggests the existence of a positive-feedback signaling pathway where levels of the ligand (VEGF) may control the levels of expression of its own receptor (flk-1) and offers a possible explanation of the heterozygous lethal phenotype in mice.11 12
The VEGF receptor gene–deletion studies also strongly suggest that critical signals provided by VEGF during vasculogenesis cannot be replaced by other endothelial cell mitogens and angiogenic factors that are likely to be present, such as acidic or basic fibroblast growth factors. Indeed, it is surprising that at such a critical step in embryonic development, ie, the establishment of a vascular network necessary to nourish the rapidly growing embryo, redundant and compensatory mechanisms do not seem to operate to ensure that formation of an essential vascular supply will occur under conditions of reduced or absent VEGF production. These findings in mice also suggest that it may be difficult to find hypomorphic alleles of VEGF in humans because they would be predicted to have severe consequences for vasculogenesis in the developing embryo.
Given the remarkable sensitivity for VEGF gene dosage during embryonic vascular development, it is not unreasonable to expect that small changes in local VEGF concentrations in adult tissues may have profound effects on angiogenesis in vivo. Thus, when one considers a strategy for gene therapy aimed at therapeutic angiogenesis using VEGF, it is conceivable that successful results could be obtained with only small changes in the local concentration of VEGF in target tissues. This brings us back to the study by Tsurumi et al reported in this issue of Circulation.1 Much of the recent emphasis on vascular gene therapy has focused on technical improvements in recombinant adenoviral vectors that will permit more efficient gene transfer to specific cell types, avoid immune system responses, and maintain high levels of gene expression for long periods of time. Certainly, for most anticipated therapeutic targets, these objectives remain important and necessary. In selected instances, however, the therapeutic utility of gene transfer may not have to wait for future achievements in these technically demanding areas.
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Ischemic Rabbit Hindlimb: A Favorable Model for Gene Transfer Using VEGF-Encoding Plasmid DNA |
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TopIntroductionVEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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The ongoing work from Isner and colleagues1 15 suggests that therapeutic angiogenesis for ischemic peripheral vascular disease could be one such clinical setting in which the necessary tools may already be at hand. Although the use of naked plasmid DNA has inherent limitations, such as the relatively low efficiency of gene transfer that results in low levels of gene expression, it does have the obvious advantage of avoiding the undesirable acute inflammatory responses prevalent with adenoviral vectors. However, in the rabbit ischemic hindlimb model used here, a combination of favorable conditions makes direct injection of VEGF plasmid DNA more likely to produce a successful outcome than if the same strategy were used in other settings or with other angiogenesis factors. First and foremost is the choice of VEGF itself. Not only is it endothelial specific in target cell profile, but hypoxia has recently been demonstrated to be a strong stimulus for upregulation of VEGF receptor gene expression.7 Therefore, in ischemic tissue, endothelial cells are likely to be more responsive to VEGF than in normal tissue, and the ability of low concentrations of exogenously delivered VEGF to effectively stimulate angiogenesis would be augmented. Thus, the low efficiency of gene transfer with direct injections of plasmid DNA may not be the limitation that it has often proved to be in other settings. The 165–amino acid form of VEGF used by Tsurumi et al1 is a secreted, heparin-binding growth factor and would likely concentrate over time around the target endothelial cells in ischemic tissue, further increasing the effective concentration of low levels of VEGF.3 In addition, VEGF promotes the physiological maturation of newly formed collateral channels and restores appropriate endothelium-derived relaxing factor/nitric oxide–dependent vasorelaxation responses initiated by the endothelium, thereby increasing blood flow through the expanding vascular bed.15 16 Moreover, hypoxic skeletal muscle has been shown to exhibit an increased uptake and expression of exogenous plasmid DNA compared with normal muscle, probably because of hypoxia-induced cell death and secondary satellite cell proliferative responses that occur within a chronically ischemic muscle bed.17 Thus, a combination of favorable circumstances makes the rabbit ischemic hindlimb model used here a particularly ideal setting for a gene delivery approach to targeted angiogenesis based on the uptake and expression of naked plasmid DNA encoding VEGF by skeletal muscle cells in the hypoxic regions.
Although the results obtained by Tsurumi et al1 with this relatively simple gene transfer method for therapeutic angiogenesis are certainly encouraging, a number of caveats remain to be taken into consideration with regard to the potential of approaches of this type for human gene therapy. First, one can expect considerable variation in the number of muscle cells that take up and express the exogenous plasmid DNA. Wolff and colleagues17 performed extensive studies to examine the effects of common variables for gene transfer with naked plasmid DNA after intramuscular injections and found that standard errors around one half of the mean were to be expected with this method. Second, unlike injections of VEGF protein itself, one cannot easily turn off production of VEGF from plasmid DNA if complications arise after gene transfer has been performed. VEGF is not only an endothelial cell mitogen and angiogenesis factor, but it also is a potent vascular hyperpermeability factor that may cause leakage of plasma constituents, including vasoconstrictors, fibrinogen, and coagulation factors, into the subendothelial layer of smooth muscle cells. The resulting vasoconstriction and/or fibrin clot formation and retraction that may occur could act to reduce blood flow through the ischemic vascular bed. Third, although the authors are careful to show that VEGF plasmid DNA cannot be detected in tissues distant from the sites of plasmid DNA injections in ischemic hindlimb, the same cannot be said about VEGF protein produced after gene transfer. There remains the concern that circulating VEGF levels may increase to the point that initiation of latent tumor growth or exacerbation of diabetic retinopathy could become clinical limitations. Furthermore, the authors do not actually show that end-point hindlimb muscle performance was improved by the levels of increased blood flow (50%) that were achieved by VEGF plasmid DNA injections. Neither do they present a compelling argument for why injections of VEGF plasmid DNA are necessarily better than injections of the recombinant VEGF protein itself, which they have shown in previous studies15 to be as effective at producing measurable angiogenic responses as shown here for plasmid DNA injections and which would be much easier to terminate if problems were to arise. Last, it remains to be seen if the VEGF plasmid DNA delivery strategy described by Tsurumi et al1 for therapeutic angiogenesis in an ischemic peripheral vascular bed will be successful in other settings.
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Summary |
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TopIntroductionVEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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Nevertheless, the simplicity of using injections of naked plasmid DNA into skeletal muscle tissue as an effective means to deliver a potent, secreted angiogenesis factor into ischemic peripheral vascular beds is both an exciting and encouraging finding. It is estimated that 150 000 patients per year require lower-limb amputations for ischemic peripheral vascular disease in the United States. The impressive progress being made toward the use of VEGF gene therapy for effective therapeutic angiogenesis in ischemic peripheral vascular disease is truly welcome news for clinicians faced with the task of providing care for those patients suffering from lower-limb vascular insufficiency.
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Acknowledgments |
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The author is supported by a grant (HL-47655) from the NHLBI and by the American Heart Association. Helpful conversations with Dr Brent A. French during the preparation of this article are gratefully acknowledged.
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Footnotes |
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The opinions in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionVEGF: An Endothelial...VEGF in Embryonic VasculogenesisIschemic Rabbit Hindlimb: A...SummaryReferences |
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1. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation. 1996;94:3281-3290.
2.
Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983-985.
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Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev.. 1992;13:18-32.
7. Tuder RM, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/flk and flt in lungs exposed to acute or chronic hypoxia. J Clin Invest.. 1995;95:1798-1807.
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Levy AP, Levy NS, Wegner S, Goldberg MA. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem.. 1995;270:13333-13340.
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12. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439-442.[Medline] [Order article via Infotrieve]
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Drake CJ, Little CD. Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization. Proc Natl Acad Sci U S A.. 1995;92:7657-7661.
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Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor. Circulation. 1995;91:2802-2809.
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